A comprehensive presentation on Chemistry of proteins with their clinical applications for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.
3. Properties of proteins
❖Definition : Proteins is derived from Greek word “ proteios which
means primary or holding first place ” .
Properties of proteins :
1. Organic compounds
2. High molecular weight
3. Rich in nitrogen
4. Important for life
5. Abundant occurrence in nature
6. Polymers of amino acids linked by peptide bonds ( CO-NH) in
specific and unique sequence which defines both their three
dimensional structure determining biological functions
4. Biochemistry of proteins
• Nitrogen content of proteins →16% by Kjeldahl method
• Nitrogen content x 6.25 = concentration of protein
• Protein are polymer of amino acids ( building blocks of proteins).
• Proteins+ HCl →hydrolysis→ L -alpha amino acids
• A1-A2-A3→(Acid /Enzyme Hydrolysis )→ A1 +A2 +A3
• 300 amino acids exist in nature ( animal ,plant and microbial system) .
• 20 standard L- amino acids commonly found in proteins of human
body = 10 essential + 10 non essential amino acids
• 61 Genetic codes for 20 standard amino acids
5. Elemental composition of proteins
Element Composition (%)
Carbon 50-55
Hydrogen 6-7
Oxygen 19-24
Nitrogen 18-19
Sulphur 0-4
phosphorous Variable (constituent of
phosphoprotein
Major components
Minor component
6. Role of metals /elements as constituent of proteins
Metal /element Constituent of Protein
Iron (Fe2+) Cytochrome oxidase
Copper (Cu2+) Ceruloplasmin
Zinc (Zn2+) DNA polymerase
Manganese (Mn2+) Superoxide dismutase
Iodine (I) Thyroid hormones
Magnesium (Mg2+) Glucokinase , Hexokinase
Phosphorous (P) Casein
8. Peptide bond formation
• Formation of peptide bond = a
covalent bond is formed
between the carboxyl group of
one amino acid and amino
group of another amino acid by
a amide linkage with removal of
a water molecule.
• Successive amino acids are
joined by peptide bonds
( -CO-NH) in proteins .
Dipeptide :two amino acids one peptide bond
9. Peptides
➢Dipeptide = 2 amino acids & one peptide bond ,tripeptide = 3 amino
acids & 2 peptide bonds , polypeptide = >10 amino acids
➢Oligopeptide: a few amino acids joined together covalently by
peptide bonds
➢Polypeptide : many amino acids joined together covalently by
peptide bonds
➢Pseudopeptide : a peptide bond is formed by carboxyl group other
than that present in the alpha position
➢Hydrolysis of peptide bonds : by proteolytic enzymes i.e. peptidases
or peptidases
10. Biologically important peptides
❖Oligopeptide: have 10 or less amino acids joined together by peptide bonds.
Biologically important peptide Type Numberofamino
acids(N)
Numberofpeptide
bonds (N-1)
Camosine and Anserine Dipeptide 2 1
Glutathione(antioxidants) Tripeptide 3 2
Thyrotropin releasing Hormone (TRH) Tripeptide 3 2
Gastrin & Secretin Pentapeptide 5 4
Enkephalin and endorphin Pentapeptide 5 4
Oxytocin Nonapeptide 9 8
Vasopressin (ADH) Nonapeptide 9 8
Bradykinin & Kallikrein Nonapeptides 9 8
Angiotensin I Decapeptide 10 9
Gramicidin S decapeptide 10 9
12. Camosine and Anserine :Biologically important dipeptides
• Camosine and Anserine : dipeptides found in human skeletal muscle
but not in cardiac muscle. Both chelate copper and enhance copper
uptake.
• Camosine : formed from peptide bond between alanine and Histidine
• Anserine : a derivative of Camosine and activates myosin ATPase
activity.
13. Glutathione: Biologically important tripeptide
❖Glutathione :
• Tripeptide ( 3amino acids & 2 peptide bonds made up of Glutamic acid ---
Cysteine---Glycine( Glutamyl- Cysteinyl- Glycine) .
• Glutamate in Glutathione is linked to Cysteine through carboxyl peptide
bond rather than carboxyl peptide bond .
• GSH is characterized by its peptide bond not attacked by peptidase .
• Glutathione may exist as the reduced(GSH) or oxidized( G-S-S-G) form and can
thus play role in oxidation –reduction reactions. In oxidized( G-S-S-G) form
two cysteine residues are linked by disulphide bonds.
• Sulfhydryl (SH) is the functional group primarily responsible for the properties
of Glutathione.
• It is found in all mammalian cells except the neurons .
• It is synthesized in two sequential steps.
14. Biosynthesis of Glutathione(Tripeptide)
Glutathione :present in RBC in large amount
Tripeptide : Glutamic acid-Cysteine-Glycine
Reduced Glutathione functions as an
antioxidant and is regenerated.
Gamma peptide bond
The gamma carboxyl group of glutamic acid
enter into peptide bond formation.
15. Functions of Glutathione
❖ Functions of Glutathione:
1. an antioxidant in RBC
a. Maintains RBC membrane integrity
b. Protects Hemoglobin from oxidation by H2O2
2. prevents oxidation of SH group containing proteins & enzymes
3. Contributes Detoxification of Organophosphate or nitro compounds
4. Functions as an antiaging agent
5. scavenger of free radicals during biological oxidation
6. helps in transport of amino acids across the cell membrane of kidney or
intestine through Meister cycle or Glutamyl cycle.
7. coenzymes of Glyoxalase in Prostaglandin Synthase
8. Plays role in prevention of Alzheimer's disease:
16. Function of Glutathione as an antioxidant in RBC
❖Function of Glutathione as an antioxidant : detoxification of H2O2 and
organic/ lipid peroxides' 2GSH (reduced ) + H2O2 ↔ GS-SG (oxidized ) +2H2O
❖Functions of reduced Glutathione in RBC
1. Maintains RBC membrane integrity (H2O2 causes lysis of RBC →Anemia) .
RBC has high concentration of reduced Glutathione. GSH protects SH group
of various proteins, decomposes the toxic hydrogen peroxide. Thus
maintains integrity of cell membrane of RBC.
2. Protects Hemoglobin from oxidation by H2O2, and keeps iron of hemoglobin
in ferrous (reduced) form.
Whenever hemoglobin is oxidized to methemoglobin (iron of
hemoglobin in ferric or oxidized ) form , reduced Glutathione (GSH )reduces it
back to hemoglobin .In this reduction , Glutathione itself gets oxidized(GS-SG).
17. Glutathione functions as an antioxidant in RBC
ReducedGlutathionefunctionsas
anantioxidantandisregenerated.
RBCwithlowerGSHaremore
susceptibletohemolysis.
Glutathione maintains RBC membrane integrity (H2O2 causes lysis of RBC →Anemia) and keeps
hemoglobin in the ferrous state . RBC has high concentration of Glutathione. GSH protects SH
group of various proteins, decomposes the toxic hydrogen peroxide. Thus maintains integrity
of cell membrane .
18. Glutathione prevents oxidation of SH group containing proteins & enzymes
❖ Proteins with SH group are functional i.e.
active state) → oxidation → Proteins with
S-S group (non- functional i.e. inactive).
❖Glutathione prevents oxidation of SH
group containing proteins & enzymes.
Donor of reducing equivalents
e.g.Wheneverhemoglobinisoxidizedtomethemoglobin(ironofhemoglobininferric oroxidized)form,
reducedGlutathione(GSH)reducesitbacktohemoglobin.Inthisreduction,Glutathioneitselfgets
oxidized(GS-SG).
19. Detoxification function of Glutathione :
❖Detoxification function of Glutathione :
Organophosphate or nitro compounds + Glutathione→ Mercapturic
acid (water soluble excretion by kidney )
20. Glutathione functions as an antiaging agent
Glutathione reduces oxidative stress by
scavenging free radicals.
Aging is associated with increased
disulfide bond formation in the SH
group containing proteins and enzymes.
21. Glutathione functions as scavenger of free radicals during biological oxidation
Biological oxidation
Type of
oxidation
Number of
electrons
Transported
towards oxygen
Product
Complete
oxidation
4 electrons water
incomplete
oxidation
2 electrons Hydrogen
peroxide
incomplete
oxidation
1 electrons Superoxide
Reducedformofglutathione–GSHwithfreesulfhydrylgroupservesassulfhydrylbufferorredoxbuffer
regulatingredoxstateofthecellbymaintaininganequilibriumbetweenreducedand oxidized state(ratioof
oxidizedtoreduced=500:1)
22. Glutathione: helps in transport of amino acids across the cell membrane
of brain, kidneys or intestine through Meister cycle and contributes absorption
of amino acids in cells.
24. Role of Glutathione in prevention of Alzheimer's disease
Alzheimer's disease: is due cellular accumulation of aggregates of misfolded proteins or
their partially degraded products . The term Prion ( proteinous infectious agents ) is coined
to collectively represent them. Reduced Glutathione prevents amyloid formation by
activating enzyme Glyoxalase and thus restores memory.
25. TRH and TSH as biologically important tripeptides
❖Thyrotropin releasing Hormone (TRH): A tripeptide with the sequence
Glu-His-Pro but Glu and Pro are modified) from Hypothalamus→
Hypothalamus
↓TRH
Pituitary gland
↓TSH
Thyroid gland
↙ ↘
T3 T4
❖ Function of Thyrotropin releasing Hormone (TRH): to release
thyrotropic hormones from anterior Pituitary gland
26. Enkephalins and endorphin (endogenous opiates): pentapeptide
❖Methionine Enkephalins : Try- Gly- Gly- Phe -Met → a pentapeptide
• a pentapeptide (made up of 5 amino acids)found in the brain
• Represent body’s own mechanisms for control of pain
• Bind to specific receptors of brain cells which can also bind to
morphine/heroin / addicting drugs and cause a similar physiological
effect (analgesia →deadening of pain sensation).
• Influence transmission in some parts of the brain.
• They are called “opioid like peptides” as they inhibit the sense of pain
(deadening of pain sensations → induce analgesia) .
❖Leucine Enkephalins : Try- Gly- Gly- Phe -Leu →a pentapeptide
27. Bradykinin & Kallikrein : biologically important nonapeptides
❖Bradykinin & Kallikrein are nonapeptides and vasodilators → decrease blood pressure.
They are formed from plasma proteins produced by snake venom enzymes.
❖Bradykinin : Arg - Pro - Pro - Gly - Phe - Ser - Pro - Phe - Arg
➢is a peptide formed by the action of proteolytic enzyme Trypsin on the 2 –globulin of
plasma . It is also formed by the action of proteolytic enzymes of snake venom on the
plasma protein .
Trypsin
2 –globulin of plasma Bradykinin(nonapeptide )
❖Functions of Bradykinin :
1. stimulate smooth muscles contraction but produces power vasodilation and fall in
arterial blood pressure.
2. causes intense peripheral and visceral pain stimulating the pain receptors
3. functions as an antioxidant
4. Hormone like peptide that inhibits inflammatory reactions
5. Hydrolyzed by Kininase
31. Oxytocin and vasopressin : biologically important nonapeptides
❖Oxytocin and Vasopressin (Anti diuretic hormone=ADH) :
1. Nonapeptide : made up of 9 amino acids
2. Secreted from posterior pituitary
❖ Amino acid sequence of Oxytocin:
S-S
Cys - Try - Ile - Gln - Asn - Cys - Pro - Leu - Gly - NH3
❖Function of Oxytocin : stimulates contraction of uterus
❖Function of vasopressin (Anti diuretic hormone=ADH) : stimulates
kidneys to retain water( anti diuretic effect) increases blood pressure
32. Oxytocin (nonapeptide) from posterior pituitary
Oxytocin (nonapeptide) from posterior pituitary stimulates uterine contraction and milk
ejection from lactating beast . Pitocin(synthetic oxytocin) is used for Induction of labor.
33. Function of Oxytocin (nonapeptide) in Induction of labor
Posterior Pituitary
Oxytocin
Uterine contraction
Labor
Number of oxytocin receptors increased
35. Vasopressin (ADH)-nonapeptide maintains osmotic pressure and water
homeostasis
Posterior pituitary →ADH → reabsorption of water by kidney → increase in
blood volume → increased blood volume→ decreased osmotic pressure
ADH →vasoconstriction → increased blood pressure
Anti diuretic action
36. Function of Angiotensin: biologically important Peptides
renin from kidney peptidase(Angiotensin converting enzyme ACE)
2 globulin Angiotensin I (decapeptide) Angiotensin II (octapeptide)
Angiotensinogen ↓
(liver) Adrenal gland
↓
Release of Aldosterone
↓
increases blood pressure
❖Function of Angiotensin II:
➢ stimulate smooth muscles contraction
➢ increases arterial blood pressure ( by constricting arterioles and increasing heart rate)
➢Promotes synthesis of a steroid hormone aldosterone that promote sodium retention
37. Angiotensin and Aldosterone maintain sodium homeostasis and blood pressure
Angiotensin converting enzyme (ACE) inhibitors like Captopril and Enalapril are used in
treatment of hypertension and congestive cardiac failure.
(ACE) inhibitors : Captopril and Enalapril
38. Gastrin & Secretin: gastro intestinal hormones (Biologically important
peptides)
Gastrin is a local hormone produced by stomach stimulated by the presence of food in
stomach . It stimulates gastric juice .
39. Gramicidin S: a circular decapeptide(10 amino acids)
D-Phe → L-Leu → L-Orn → L-Val → L-Pro
L-Pro L-Val L-Orn L-Leu D-Phe
❖ Gramicidin S :
➢ is an antibiotic .
➢ contains two residues of a D –amino acids (D-Phe).
✓Orn : symbol for Ornithine, an amino acid that does not occur in proteins.
The arrows lead from the amino terminal
end towards carboxyl-terminal residue.
40. Gramicidin S , Bacitracin, Actinomycin & Tyrocidine: Biologically important peptides
(peptide antibodies)
Gramicidin S : a decapeptide(10 amino acids) is in a circular form and contains D –
phenylalanine( usual protein contains L –amino acids) . It is an antibiotic produced by Bacillus
brevis .
41. Commercial /Artificial synthesis peptides
❖Commercial /Artificial synthesis peptides by solid phase technique :
• This is achieved by fixing the carboxy group at C- terminal end amino acid to
insoluble polystyrene beads of resin and other amino acids are added
sequentially.
• Reaction occur between an activated carboxy group such as an acid chloride of
one amino acid and the amino group of another e.g. Cysteine acid chloride and
Lysine .After the peptide bond formation ,the blocking group is removed,leaving
desired peptide.
• It facilitates washing and purification of peptide .
• This process is simple and rapid .
• It was introduced by Brace Merrifield (Nobel 1964).
• Insulin was the first major protein chemically synthesized by Katsoyanis 1964.
42. Application of artificially synthesized peptides
❖Application of artificially synthesized peptides:
1. To check whether the sequence analysis is correct or not
2. To map the active site / biologically important area after alteration of
primary structure of a peptide by one or two amino acids
3. To avail pure preparations for medical or diagnostic purpose. Artificial
synthesis antigenic part of protein for detection of antibodies in serum of
patient using ELISA is cheaper and faster. Preparations of large quantity of
antigen from virus (e.g. HIV) is less tedious and safer than isolating from
biological sources. Pure antigen is to be coated in the test tube in ELISA
technique .
4. Commercial synthesis of Insulin for therapeutic purpose( Insulin first major
protein chemically synthesized by Katsoyanis 1964)
43. Aspartame : synthetic peptide
❖Aspartame/ nutra sweet :
• Artificial dipeptide :(L-Aspartyl –Phenylalaninyl Methyl Ester =Aspartic acid +
Phenylalanine)
• Artificial sweeter : 200 times sweeter than sucrose and used as a low calorie
artificial sweetener in soft drink industry.
• Content of Phenylalanine of makes Aspartame/ nutra sweet , it unsuitable
for patients with phenylketonuria.
46. Static and dynamic functions of proteins
1. Proteins involved in static (structural ) functions :
a. Collagen
b. Alpha keratin
c. Elastin
d. Bone matrix
2. Proteins involved in dynamic functions:
a) Hormones f)muscle contraction proteins
b) Enzymes g) genetic control proteins
c) Blood clotting factors h) proteins involved in respiration
d) Immunoglobulins
e) Membrane proteins
47. Functions of proteins
Proteins
1. Play role in cell structure and functions.
2. Constitute 17 % of body weight.
3. Necessary for the formation of the membrane , muscle and
connective tissue.
4. Transport compounds across cell membrane.
5. Confer immunity against viral and bacterial infections.
6. Functions as buffer to maintain pH of the cell
7. Needed for growth and repair of tissues.
8. All enzymes are proteins.
9. Many hormones are proteins.
48. Transport proteins
Transport proteins : in blood /plasma bind and carry specific molecules or ions
from one organ to another.
Transport proteins Functions of Transport proteins
Hemoglobin Binds to oxygen in lung and carries it to the peripheral tissues
Transferrin(-globulin) Transport of iron
Ceruloplasmin (2- globulin) Transport of Copper in human serum
Lipoprotein HDL (1- globulin) Cholesterol from extrahepatic tissue to the liver for excretion
Lipoprotein LDL (-globulin) Cholesterol from liver to peripheral tissue
Transport proteins in plasma membrane /
intracellular membranes
Bind to glucose ,amino acids or other substances and transport
them across the membrane
Retinal binding protein- RBP (1- globulin) Vitamin A
Transcortin Cortisol hormone
Thyroxine binding protein-TBG (2- globulin) Thyroxine hormone
Albumin Poorly soluble substances : fatty acids ,bilirubin ,Calcium ,steroid
hormones , Sulfonamide ,Penicillin ,Dicumarol ,Aspirin ,Digoxin
49. Storage proteins
Storage proteins Functions for Storage of
Apoferritin Iron in form of ferritin
Myoglobin Oxygen in muscles
Albumin Amino acids –protein energy
50. Biological functions of proteins:1
Class of proteins Example of proteins Functions
Contractile proteins Actin and myosin Present in thin and thick filaments
respectively
Defense /Protective
proteins
Immunoglobulins Confers immunity against infection
Fibrinogen Forms fibrin blood clot
Enzymes Hexokinase Phosphorylates glucose
Regulatory
proteins/Hormones
insulin, Glucagon regulation of glucose metabolism
Storage proteins Ferritin Storage of iron in liver , spleen and
bone marrow
Structural proteins Collagen Present in fibrous connective tissue
( bone, tendon , cartilage)
Proteins as buffers Plasma proteins Buffering in plasma
51. Biological functions of proteins:2
Class of proteins Example of
proteins
Functions
Transport proteins Hemoglobin Transport of O2 to tissue and CO2
from tissue
Albumin Transport of fatty acids , bilirubin
Transferrin Transport of iron in blood
Lipoproteins Transport of lipids in blood
Protein as toxins Clostridium,
Botulinum toxin
Cause of bacterial food poisoning
Protein as antivitamin Avidin of egg white Binds vitamin biotin
These are present in different biological systems .
52. Classification of proteins based on Nutritional value
Complete or first
class proteins
Incomplete proteins Partially Incomplete or
poor proteins
1 has all 10 essential
amino acids
1 essential amino acids
missing
Partially lacking one or
more essential amino acids
2 Promote growth
satisfactory in
children when
supplied in diet
Cannot Promote body
growth in children
May be able to sustain the
original body weight in
adults
Promote moderate
growth and will not even
sustain the original body
weight in adults
3 e.g. Casein of milk,
Egg albumin
e.g. Gelatin (Trp absent)
Zein (Trp/Lys absent)
Pulses(lack Met)
Cereals (Lys absent )
e.g. Wheat ,rice (Trp, Lys
absent)
53. Classification of proteins based on Chemical nature composition
Class Chemical nature
Simple proteins Composed of amino acids only e.g. serum /egg Albumin,
Keratin ( hair), Lactalbumin, Myosin of muscle
Conjugated
proteins
Composed of amino acids only and prosthetic (additional)
group (non-protein part)e .g. Hemoglobin , Casein,
Rhodopsin, Ferritin , glycoproteins , lipoproteins ,
nucleoproteins
Derived/
Denatured
proteins
Derived or denatured products of high molecular weight
Simple & Conjugated proteins by partial hydrolysis e.g.
peptone , gelatin from collagen
Progressive hydrolysis of proteins results in smaller and
smaller chains : protein → peptone → peptides→ amino
acids
54. Classification of proteins based on the constituents
❖A protein molecule contains one or more polypeptide.
Class of proteins Constituents Example
Simple proteins Contain only polypeptide Albumin
Complex proteins Contain in addition to
polypeptides , a non-amino acid
component called prosthetic group
such as
Lipids in Lipoproteins
Heme in hemoprotein
Metals in metalloprotein
Phosphategroupinphosphoprotein
LDL
Hemoglobin
Transferrin
Casein
55. Classification of Simple proteins based on shape
❖Classification of Simple proteins based on shape : Globular proteins
and Fibrous proteins
Criteria Globular proteins Fibrous proteins
Shape Spherical/oval Long Fibrous-elongated
or needle shaped
Solubility Water soluble Water insoluble
Digestibility Digestible Resistant to digestion
Examples e.g. Hemoglobin, Albumin
,globulins , protamine ,
many enzymes
e.g. Keratin of hair,
Myosin- contractile
protein from muscles ,
Collagen of connective
tissue
56. Properties of Scleroproteins and Spheroproteins : Classification of proteins based
on shape
Class of proteins Properties of proteins examples
Fibrous proteins/
Scleroproteins
high molecular weight , insoluble
in aqueous solution, found as
extended linear fibers long and
thin with
axial ratio (length/ breath >10)
Collagen- found in
cartilage /tendon
Elastin- found in elastic
tissue such as tendon,
arteries
Keratin–hair , skin , nail
Globular proteins /
Spheroproteins
Ovoid/ Spherical shape ,low
molecular weight ,soluble in
aqueous solution, tightly coiled/
folded three dimensionally,
axial ratio (length/ breath <10)
Hemoglobin, Albumin ,
Globulin ,many
enzymes, Protamine ,
Histones ,Actin,
Troponin
59. Functions of Fibrous proteins
Fibrous proteins with
structural function
Functions of Fibrous proteins (constituent of )
Collagen Fibrous Connective tissue(tendon ,bone ,cartilage)
Elastin Elastic Connective tissue (arteries /tendon/
ligaments )
-Keratin(found in
mammals almost
entirely with - helical
structure)
Hair /nail /horn
-Keratin(made up of -
sheets occur in feathers)
feather
61. Elongated protein :Human fibrinogen
Elongated protein molecules increase the viscosity of the solution.
62. Constituent amino acids of triple stranded helix Structure of Collagen
• ScvConstituent amino
acids of Collagen
% of total amino acids
Glycine 33
Proline and hydroxy
proline
21
Lysine and hydroxy
Lysine
3
Alanine 11
Arginine 5
Cysteine and
Tryptophan
absent
Scurvy:vitaminCdeficiency→failure ofhydroxylationofProlineandLysineleadstoreducedhydrogen
bonding→weaknessofcollagen→Brittlebonedisease:mutation→replacementofcentralGlycine
63. Properties of Globular proteins
Globular Protein Properties of Globular proteins
Albumin Soluble in water, coagulated by heat, molecular weight 69000,deficient in
Glycine
Globulins insoluble in pure water, soluble in dilute salt solution, coagulated by heat
Glutelins Plant proteins ,Soluble in dilute acids and alkalies , insoluble in water
Histones Soluble in water, not coagulated by heat ,rich in basic amino acids (H, A ,L)
Prolamins insoluble in pure water neutral solvents and absolute alcohol, soluble in
70-80 % alcohol ,rich in Proline and lacks Lysine
Protamine Soluble in water and dilute acids, not coagulated by heat, contain large
number Arginine and lysine residues .They are strongly basic and can
combine with other acidic proteins ,occur in tissues with nucleic acidic
Lectins Precipitatedby30-60%ammoniumsulphate,havinghighaffinitytosugargroup
Scleroproteins insoluble in pure water, salt solution ,organic solvents and soluble only in
hot strong acids
64. Functions of Globular proteins
Globular Protein Functions of Globular proteins
Albumin Nutritive(serum albumin ,ovalbumin , Lactalbumin ),transport of fatty acids,
bilirubin ,steroid hormones
Globulins Immunoglobulin ,egg yolk globulin, legumin of peas,serum globulin , myosin
Globin Along with Histone
Glutelins Glutelin (wheat ), Oryzenin (rice)
Histones salt combination with acidic DNA (nucleosomes) and RNA, globin of
hemoglobin
Prolamins Zein ( corn) ,Gliadin (wheat ),hordein from barley
Protamine Combine with acidic proteins & nucleic acid , e.g. nucleoprotein of sperm ,
Protamine Zinc Insulate (commercial preparation )
Lectins Attachmenttonormalandcancercellisdifferent(helpsintheir differentiation)
Scleroproteins Constituentofsupportingtissuecartilage,tendon,keratinofhair,nail,horn,hoof
65. Prosthetic group of Conjugated proteins
Conjugated proteins Prosthetic group of Conjugated proteins
Mucoprotein Carbohydrate- different monosaccharides and their derivatives > 4%
(mucin of saliva ,ovomucoid, glycosaminoglycan )
Glycoprotein Carbohydrate < 4% ( immunoglobulins , TSH,FSH, LH )
Lipoprotein Lipids(chylomicrons,LDL,HDL,VLDL)Triglyceride,Phospholipid,freeandester
cholesterol withApolipoprotein(constituentofacellmembrane)
Nucleoprotein Nucleic acids - DNA ,RNA ( nucleo -histones/nucleo -protamine)
Phosphoprotein Phosphoric acid (Casein of milk ,vitelline of egg yolk )
Chromoprotein Heme(Hemoglobin,Cytochromes,catalase,peroxidase),FMN/FAD(flavoproteins)
Metalloprotein Cu2+ (Ceruloplasmin) , Zn 2+ (Carbonic anhydrase ,DNA polymerase )
Rhodopsin 11 cis-retinal with protein opsin( visual pigment of retina)
Ferritin Non-heme Iron (Fe +++)with protein part –apoferritin
Hemoglobin Heme( contains ferrous Iron- (Fe ++)-with protein part globin
66. Classification of Derived Proteins
Primary Derived Proteins
(denatured proteins)
Secondary Derived Proteins
Derived from proteins by agents
such as heat , acids , alkalis
which cause only slight changes
in protein molecule and its
properties without hydrolytic
cleavage of peptide bond
Derived in the progressive
hydrolytic cleavage of the peptide
bonds of metaproteins
(coagulated proteins)into
progressive smaller molecules
e.g. Protean , Metaproteins Proteoses→peptones→peptides
67. Examples of Derived Proteins
Derived Proteins Examples
Coagulated proteins cooked proteins (egg albumin)
Protean (insoluble in water )-formed after action of
acids/alkalis/ enzymes on proteins
e.g. fibrinogen→ fibrin, myosin → myosan
Metaproteins By further action of acids and alkalies on
Proteans e.g. Acid and alkali albuminates
Proteoses ,Peptones,
Peptides
hydrolytic products of proteins
68. Successive stages complete hydrolytic decomposition of a
native protein molecule into amino acids
Protein
Proteans
Metaproteins
Proteoses
Peptones
Peptides
Aminoacids
Primary Derivatives
Secondary Derivatives
69. Classification of proteins based solubility
Solubility Examples
In water Serum Albumin ,egg albumin , lactalbumin
In dilute alkali solutions Serum globulin ,ovoglobulin
In dilute acid solutions Histones(nucleoprotein rich basic amino acids and
have high molecular weight . It is protein part of
Hemoglobin )
In dilute acid solutions Protamine(richbasicaminoacidsArginineandhavelow
molecularweight)e.g.Salmineofsalmonsperm
Insoluble in water and
salt solutions
Scleroproteins (connective tissue proteins e.g.
Collagens , Elastin , Casein
Insoluble in water and
soluble in 70% alcohol
Prolamins (rich in Proline) e. g. Gliadin, Zein
70. Clinical applications of Lectins
Source of Lectin Clinical applications of Lectins
Lectin from Dolichols biflorus Agglutinate human blood group A1 RBC
Phytohemagglutinin(PHA)-a
lectin from Phaseolus vulgaris
(red kidney bean)
Agglutinate all human RBCs and WBCs
Concanavalin – A(Con A) – a
lectin from legume
will specifically attach to Mannose and
Glucose. The attachment of this lectin on
normal and cancer cells will be different. This
property is sometimes utilized to
differentiate cancer cells.
72. Physical properties of proteins
1. Solubility :protein solution exhibit colloidal properties (huge size ,not
true solution) and therefore scatter light. Protein solution exert osmotic
pressure. Osmotic pressure of plasma protein is clinically important .
2. Molecular weight : average molecular weight of amino acid: 110
3. Shape : globular (Insulin ),oval (= globular proteins e.g. Albumin ),Fibrous
(= Scleroproteins e.g. Fibrinogen , keratin , collagen )
Generally , the polypeptide chains are linear. Very rarely, proteins may be a
branched form or circular form (Gramicidin ) .
The branching points in the polypeptide chain may be produced by
interchain disulphide bridges (the covalent disulphide bonds between
different polypeptide chains in the same protein) or portion of the same
polypeptide chain (intrachain) . They are part of primary structure.
4 . Viscosity : expressed since proteins impart tendency of a liquid to flow
5. Isoelectric point: made up of amino acids ,the pI of constituent amino
acids will influence the pI of protein .
6. Hydration of proteins : protein swell when brought in contact with water
73. Solubility : Physical properties of proteins: 1.
1. Solubility : Globular proteins (e.g. Albumin) have higher solubility
than elongated fibrous proteins(e.g. Elastin) .Smaller Globular
proteins are more soluble than lager Globular proteins .
74. Osmotic pressure of plasma protein
❖Properties of Colloidal proteins: particle size 1-100 m in dimension,
heavier than water and therefore sink in water in Colloidal solution.
❖Crystalloid / true solution : particle size < 1 m
❖Colloidal proteins exert osmotic pressure .Osmotic pressure generated
by plasma proteins is often called the Colloidal Osmotic pressure or
Osmotic pressure of plasma .
❖Osmotic pressure of plasma protein concertation of plasma protein
(directly proportional / directly related )
❖Osmotic pressure of plasma protein 1/ molecular weight
(inversely proportional / inversely related )
❖Plasma albumin : contributes 75-80% of Osmotic pressure of plasma
because of its lower molecular weight (although it represents more
than half of plasma protein)
75. Edema due to hypoalbuminemia : Clinical applications of osmolality of fluid
Edema due to hypoalbuminemia : Disorders such as kwashiorkor and
glomerular nephritis are associated with lowered plasma albumin
concentration and edema . Edema is caused by reduced osmotic pressure
of plasma leading to accumulation of excess fluid in tissue spaces.
76. Colloidal osmotic pressure of plasma and Edema
Osmotic pressure of plasma protein is clinically important in maintaining blood volume
77. Molecular weight of proteins : Physical properties of proteins:2
Proteins Molecular weight (Dalton ) of proteins
Insulin 5700
Hemoglobin 68000
Albumin 69000
Gamma-Globulins 160000
Rabbit papilloma virus protein 47000000
Fibrinogen → factor I 340,000
Prothrombin → factor II 720,000
Thromboplastin(Tissue factor)→ factor III 370,000
AntihemophilicfactorA,Antihemophilicglobulin(AHG)
→factorVIII
330,000
Christmasfactor,AntihemophilicfactorB,Plasma
Thromboplastincomponent(PTC)→factorIX
Stuart–power→factorX
56,000
Blood
coagulation
factors in
human
78. Viscosity : Physical properties of proteins:4
❖Viscosity :expresses the tendency of liquid to flow .
❖Factors affecting viscosity :
1. Molecular weight of the proteins : higher viscosity is found with lager
molecules than smaller molecules
2. Shape of protein molecules : higher viscosity is found with elongated
molecules than with spherical molecules. Fibrous proteins cause higher
viscosity than globular proteins .
3. Globulins impart greater viscosity than Albumin because of its bigger size.
79. Amphotericnature/IsoelectricpH:Physicalpropertiesofproteins:5
• The amino acid composition will determine the isoelectric pH (pI) of protein.
Amphotericnature: ionizable groups present at the ends of the polypeptide
( free NH2 ,COOH) and ionizable groups of the side chains of the amino acids of
polypeptide confer an amphoteric character of on proteins .
• The alpha amino group of N- terminal amino acid and carboxy group of C-
terminal amino acid are not utilized for peptide bond formation and hence are
ionizable. All other ionizable groups present in protein will influence the
isoelectric pH (pI) of protein .
Status of pH of solution wrt pI proteins are
acidic side of pI cations
alkaline side of pI anions
80. Acidic & Basic proteins
Acidic proteins Basic proteins
E Lys + E Arg
E Glu + E Asp
E Lys + E Arg
E Glu + E Asp
= < 1 = > 1
81. Isoelectric pH of proteins
❖At physiological p H ( 7.4 ) proteins exist as charged molecules assist Donan equilibrium.
Protein PI Charge on proteins at physiological pH( 7.4)
Pepsin 1.1 negative
Casein/egg albumin 4.6 negative
Human albumin 4.7 negative
Urease 5.0 negative
Human Insulin 5.4 negative
Human globulin 6.6 negative
Human Hemoglobin 6.7 negative
Myoglobin 7.0 negative
Chymotrypsinogen 9.5 Positive
Cytochrome C 10.7 positive
Lysozyme 11.0 Positive
82. Principle and applications of Isoelectric focusing to determine PI value of proteins
❖Principle of Isoelectric focusing to determine PI value of proteins : is
primarily based on the immobilization of the molecule at isoelectric pH during
electrophoresis .
➢Gel contains a substances called ampholyte which creates a stable pH
gradient in the gel.
➢Since the diffusion is offset by the electrical field , the bands do not broaden
as in other separation method.
❖Applications of Isoelectric focusing :
1. separation of serum proteins (separated to as many as 40 bands)
2. conveniently used for the purification of proteins
3. Tremendously powerful method and is capable of resolving proteins that
differ in pH by as little as 0.01 pH units
83. Techniqueof Isoelectric focusing to determine PI value of proteins:1
For the proteins in the solution , there is a pH at which the number of anions
formed is exactly equal to the number of cations . This pH is called the
isoelectric pH ( pI ) of that protein and exist as Zwitterion.
84. TechniqueofIsoelectricfocusingtodeterminePIvalueofproteins:2
Set up stable pH gradients in a gel covering the pH range to include the
isoelectric points of the components in a mixture of proteins
Start Electrophoresis for separation of proteins
Migrationoftheproteinmoleculestopositioncorrespondingtotheirisoelectricpoints
Immobilization of proteins at pI and formation of sharp stationary bands
Staining of gel blocks
Identification of proteins based on pI value
85. Importance of isoelectric pH of proteins
❖isoelectric pH : that pH at which the protein has equal number of positive
and negative charges ( i.e. no net charge). All ionizable groups present in the
proteins influence the pI of the proteins.
❖Proteins at pI exhibit minimum :
a. Buffering capacity
b. Viscosity
c. solubility
❖Proteins tend to aggregate and precipitate at their isoelectric pH . As a result,
they exhibit maximum precipitation and the least solubility.
❖At pH values above the isoelectric pH , they carry a net negative charge and
migrate to anode (+ vely charged electrode) . At pH values below the
isoelectric pH , they carry a net positive charge and migrate to cathode (- vely
charged electrode) . Proteins do not move under electrical field at pI .
❖Application : this property of protein has application in the separation of
protein from mixture by electrophoresis . Hence ,for the electrophoretic
separation of plasma proteins , one has to select a pH different from the
isoelectric pH .
86. Application of Isoelectric pH of proteins in electrophoretic separation
Application of Isoelectric pH of proteins in electrophoretic separation :
❖At pH 8.6 ( Barbitone buffer ), Albumin (PI =4.7) & Globulin ( PI = 6.7 )carry
negative charge and move towards anode during Protein electrophoresis
based on molecular weight .
Criteria pH < pI pH = pI pH > pI
Charge on amino
acids
Positive charge Neutral Negative charge
Electrophoretic
mobility
Move towards
cathode(cation)
Isoelectric
focusing
Move towards anode
(anion)
87. Application of Isoelectric pH of proteins in staining of cells
for diagnosis
Type of dye Example Reaction involved in staining
Acidic Eosin Eosin→ H+ + dye
H+ + protein → protein-NH3
+
(protein cation –red colored )
Basic Hematoxylin,
Methylene blue
Hematoxylin/ Methylene blue →
OH
- + dye
OH
- + protein → protein-COO-
(protein anion –blue colored )
Thus, the staining characteristic of a protein is determined by Isoelectric
pH of the concerned protein.
88. Hydration of proteins : Physical properties of proteins:6
6. Hydration of proteins : proteins when brought in contact with
water, absorb water and swell up.
Water envelope or solvation layer: a relatively immobile shell-like
layer of water, around each protein particle in aqueous medium
Polar groups responsible for Water envelope : COOH , NH2, OH of
protein
Mechanism of hydration of protein molecule: polar groups of
proteins bind to the molecules of water by hydrogen bonds to hold
considerable amount of water around each protein particle in an
aqueous medium
Application:proteinscanbeprecipitatedbydehydrationusingelectrolytes
andalcoholastheseagentscompeteforthewaterofhydration
90. Principle of Precipitation reaction of proteins
Principle Precipitation Reactions of proteins :
❖ The stability of proteins depends mainly on charge and hydration of protein
molecule .
❖ Polar groups of proteins ( -NH2 , COOH , OH group) tend to attract water
molecules around them to produce shell of hydration .
❖ Therefore, proteins can be precipitated either by removal of water layer by
dehydration, denaturation ,adjusting isoelectric pH ,by neutralization of
charge on present on the protein molecule .
91. Precipitation of proteins
1. using mineral acids
2.Achieving Isoelectric pH
3.Salting out
3.using Heavy metals
4.using Alkaloid reagents
5.using Organic solvents
6.Heat coagulation
❖ Proteins are precipitated by
92. Heller’s Test :Precipitation reaction of proteins
Heller’s Test Observation Inference
2ml of concentrated
nitric acid + 0.2ml OS
from side of test tube
White ring at junction
of two layers
Concentrated mineral
acids like HNO3 ,H2SO4
& HCl causes
denaturation of
proteins .
I. Precipitation of proteins using mineral acids
❖ Heller’s Test
93. Heller’s Test for detection of proteinuria
con❖ConditionsassociatedwithAlbuminuria:
1. Nephrotic syndrome
2. Inflammation of urinary tract
94. Proteins are precipitated by achieving isoelectric pH
• Proteins are least soluble at their isoelectric pH. Some proteins are
precipitated immediately when adjusted to their isoelectric pH.
Milk curdled
Lactic acid production by fermenting bacteria
pHofmilkloweredtoisoelectricpH-4.6ofCasein(milkprotein)
Precipitation of casein ( paneer formation)
Flocculentprecipitatere-dissolvesinhighlyacidicoralkalinesolution
95. Precipitationofproteinsbysaltingout (usingAmmoniumsulphate/Sodiumsulphatepowder)
Test Observation Inference
3ml of OS + solid
Ammonium sulphate
(powder )
White precipitate Concentrated salt solution
causes precipitation of proteins
by dehydration- shell of water
removed (Salting out).
Salting in : proteins in soluble state in water
Salting out : precipitation of proteins- out of water due to protein –protein
interaction
96. Precipitation of proteins by salts (using ammonium sulphate powder)
Full saturation Half saturation
1. Solid ammonium sulphate Salt/
powder used for precipitation
Saturated ammonium sulphate
solution used for precipitation
2. High &low molecular weight proteins
get precipitated depending upon
concentration of salt .
High molecular weight proteins get
easily precipitated. High molecular
weight proteins require less salt
than low molecular weight proteins.
3. Albumin are precipitated at full
saturation of ammonium sulphate or
28% Na –sulphate.
Globulins are precipitated at half
saturation of ammonium sulphate
or 22% Na -sulphate .
97. Schematic diagram for Full saturation and half saturation for fractionation
Full saturation : Half saturation:
High(Globulins) & low (Albumin )
molecularweightproteinsgetprecipitated
Low molecular weight
proteins in the
supernatant
e.g. Albumin with mw
60000
High molecular
Weight proteins
get precipitated
e.g. Globulins
with mw
180000
Application:Fullsaturationandhalfsaturationareuseful forfractionationofproteins
frommixture(e.g.separatingalbuminandglobulinfromserumproteins).
98. Precipitation of proteins by heavy metals
Test Observation Inference
3ml of OS + 2drops of 2%
sodium carbonate solution
(alkaline medium )+ 1drop
of lead acetate solution
White precipitate In alkaline medium ( on the
alkaline side of pI), the
proteins acquire a negative
charge (anion). This is
neutralized by positive charge
of heavy metals like lead- Pb
,mercury-Hg & iron- Fe which
cause precipitation. Insoluble
metal proteinates are
formed.
Therapeutic application of Precipitation of proteins by heavy metals : Lead ,Copper , Zinc,
Mercury and Cadmium are toxic as they tend to precipitate normal proteins of
gastrointestinal wall. Raw egg is sometimes used as an antidote for Mercury poisoning.
99. Therapeutic and Analytical applications of precipitation of proteins by
heavy metals
❖Analytical application of Precipitation of proteins by heavy metals:
To precipitate plasma proteins in the Nelson-Somogyi method for estimation of
blood glucose . Ba(OH)2 and ZnSO4 solution are mixed with the blood . Ba(OH)2
makes the alkaline media than pI of the plasma protein and becomes anionic
which bind with Zn 2+ to get precipitate as Zn – proteinates.
❖Therapeutic application of Precipitation of proteins by heavy metals : Lead,
Copper , Zinc, Mercury and Cadmium are toxic as they tend to precipitate
normal proteins of gastrointestinal wall. Raw egg is sometimes used as an
antidote for Mercury poisoning. Mercury will precipitate egg albumin rather
than proteins of Gastrointestinal wall.
100. Precipitation of proteins by alkaloid reagents
Alkaloid reagents like tungstic acid, Trichloro acetic acid , Sulphosalicylic acid,
Phospho tungstic acid, tannic acid & Esbach's reagent ( picric acid) precipitates
proteins in acidic medium.
Test Observation Inference
3ml of OS + 5drops of Trichloro
acetic acid solution
➢ Acid lowers pH medium
White precipitate
(flocculent precipitate)
In acidic medium(acidic side of pI),
the proteins acquire a positive
charge(cation). This is neutralized
by negative charge alkaloid
reagents.
2ml of OS + 3-4drops of 20 %
sulphosalicylic acid solution
➢ Acid lowers pH of medium
White precipitate In acidic medium ,the proteins
acquire a positive charge (cation).
This is neutralized by negative
charge alkaloid reagents .
101. Applicationsof Precipitation of proteins by alkaloid reagents
❖Applications of Precipitation of proteins by alkaloid reagents :
1.Tanning in leather processing is based on the protein precipitating effect of
Tannic acid.
2. Esbach's reagent , Trichloro acetic acid , Sulphosalicylic acid and Phospho
tungstic acid are used in qualitative/ quantitative analysis of proteins from
biological fluids .
3. To precipitate plasma proteins for estimating blood sugar by Folin-Wu
method .2/3 N H2SO4 and 10% Na tungstate ae added to blood . H2SO4
provides more acidic media than the isoelectric pH of plasma proteins. Protein
cations formed consequently bind with tungstate anion to get precipitated as
protein tungstate .
102. Precipitation of proteins by organic solvents -alcohol
Test Observation Inference
2ml of OS + 1ml of
absolute alcohol
White precipitate Alcohol (organic solvent)
precipitates proteins by
removing water shell
around protein molecule
(dehydration)leading to
denaturation.
Application of Precipitation of proteins by alcohol : alcohol is used as a
disinfectant, it causes denaturation of cell wall proteins of bacteria ,fungi and
viruses.
103. Precipitation of proteins by Heat coagulation
Heat coagulation Test Observation Inference
Take 1/3rd test tube of
OS→ Boil the upper
portion of the solution
,add 1% acetic acid to
adjust pH ,boil again
coagulum develops .The
lower layer serves as
control which remains
clear.
Coagulum formed When a protein is heated, its
physical, chemical & biological
properties are changed due breaking
of certain bonds & results in
confirmation of its molecule . The
process is known as denaturation.
Denaturation by salt precipitation
may be reversible, while that by heat
coagulation is an irreversible type of
denaturation.
104. Coagulation of proteins by heat
❖Coagulation of proteins by heat:
1. Proteins + heated at isoelectric point → heat coagulation(coagulum – thick
floating conglomerates )
2. It is a irreversible denaturation e.g. coagulated egg white of boiled egg
3. Application of heat and Acetic acid test : commonly employed detection
of proteins(albumin) in urine(proteinuria/ albuminuria)
107. Denaturation of proteins
• Since the conformation a protein is solely dependent on weak valence forces,
it can be disturbed by variety of physical and chemical agents . This process is
known as Denaturation .
• Denaturation involves breaking of secondary ,tertiary and quaternary
(hydrogen bonds, ionic bonds, hydrophobic bonds that stabilize the structure)
without breaking primary structure -no hydrolysis of peptide bonds(the
covalent bonds).
Native proteins- organized & active
Denaturation
Denatured proteins and inactive
•5 •66
•45
•3
•5-SH
•31-SH
•SH
•66SH
108. Denaturation of proteins
❖Native proteins : organized & active
❖Denatured proteins :
1. disorganized (unfolding of natural coils of native protein ) & inactive
2. Loss of secondary ,tertiary & quaternary structure ( hydrogen bonds, ionic bonds,
hydrophobic bonds that stabilize the structure to maintain its conformation in
space referred as a native state ) without breaking primary structure -no hydrolysis
of peptide bonds(the covalent bonds)
3. change of physical (increased viscosity and deceased diffusion ),chemical
(decreased solubility and increased perceptibility) & biological properties
(enzymatic activity, hormonal property and antigen –antibody interaction)
4. Denatured proteins : are digested easily (exposure of peptide bonds on surface
therefore become more accessible to action proteolytic enzymes )→ increased
digestibility
5. e.g. cooked meat, boiled egg, milk ,paneer etc.
6. The average free energy change =125 Kcal/mol (=the disruption of 3 or 4
hydrogen bonds→ proteins are characterized by a narrow range of thermodynamic
stability ) .
109. Significance of Denaturation of proteins
❖Clinical application of Denaturation of proteins :
1. Denaturation of proteins is used in biochemical blood analysis to eliminate
the proteins of blood . Deproteinization of blood is essential step as
proteins interfere biochemical color developing reactions and lead to
erroneous results.
2. Digestibility of native protein is increased on denaturation by gastric HCl or
heat on cooking .Denaturation of protein causes unfolding of native
polypeptide coil so that hidden peptide bonds are exposed to the action of
proteolytic enzymes in the gut. It also increases reactivity of certain groups.
110. Agents of Denaturation of proteins
Physical agents of Denaturation of
proteins
1.Heat
2. X-rays
3.Ultra violet and Gamma radiation
4. Violent shaking
5. Freezing and thawing
6.Pressure/ vigorous shaking/
grinding
7. Ultra sound
Chemical agents of Denaturation of
proteins
1. Acids
2. Alkali
3.Organicsolvents(Ether,Alcohol,acetone )
4.Alkaloid reagent (TCA ,
Sulphosalicylic acid, Tungstic acid)
5.Salts/detergent/guanidiumchloride
6.Heavy metals (Hg2+,Pb2+,Cu2+,Ag2+)
7.High concentration of Urea
8. Salicylates 9.beta mercaptoethanol
111. Comparison of Native proteins and Denaturation of proteins
Native proteins Denatured Proteins
with intact native primary,
secondary ,tertiary & quaternary
structure
primary structure retained
Biologically active Biologically inactive
Soluble in water insoluble in water
viscous Viscosity increases & surface
tension decreases
with intact hydrogen , S-S bonds Loss of hydrogen , S-S bonds
Crystallization feasible cannot be crystallized
112. Renaturation of proteins
❖Renaturation (reversible denaturation)of proteins: may be in rare cases be
reversible , in which case the protein refolds into its original native structure
(original tertiary structure)when the denaturing agent is removed.
❖Examples of Renaturation (reversible denaturation)of proteins :
1. Hemoglobin (Hb )+Salicylates → denatured protein hemoglobin
Denatured hemoglobin → removal of salicylates by dialysis →Renaturation of
Hemoglobin
2. Immunoglobulin + urea/ beta mercaptoethanol→ subunit of Immunoglobulin
chains dissociated as disulphide bonds are broken and polypeptide chain uncoils
Denatured Immunoglobulin → removal of urea by dialysis →Renaturation of
immunoglobulin ( slow re-oxidation to native protein)
3. Ribonuclease + urea→ denatured ribonuclease
Denatured ribonuclease → removal of urea by dialysis →Renaturation of
ribonuclease
❖Albumin cannot be renatured by removing the physical agent( heat).
113. Clinical applications of precipitation of proteins
1. Heller’s test : used for detection of albumin in urine(albuminuria)
2. Precipitation of proteins by alkaloid reagents - Trichloro acetic acid
solution/ sulphosalicylic acid used for qualitative & quantitative analysis of
proteins in biological fluids ,urine ,CSF.
3. Precipitation of proteins by Ammonium sulphate solution used for
purification of proteins & differential fractionation e.g. Albumin & Globulin
4. Milk + Lactic acid → at 4.6 pH → PANEER
5. Albumin as an antidote during treatment of heavy metal poisoning
115. Flocculation
❖Flocculation
1. Denatured albumin/proteins is soluble at extremes of pH .
2. Maximum precipitation occurs at pI of the protein i.e. albumin as
floccules (flocculation)
3. Denatured protein floccules, is reversible and soluble at extremes of
pH.
4. When floccules at pI heated further ,it becomes dense “coagulum”
which is irreversible , and not soluble at extremes of pH.
116. Schematic diagram of Denaturation, Flocculation and coagulation formation for Albumin
Acidic pH pH< pI
Native Protein(soluble)
Protein (denatured)-insoluble
Coagulum
Heat
Heat
Protein(denatured)-Soluble
pH<pl Protein Protein
(denatured) (denatured)
Flocculation soluble
pH=pl pH>pl
Alkali added OH-
Acid added H+
Alkali added OH -
Acid added H+
117.
118. Color reactions of proteins & amino acids
Test FunctionalgroupresponsibleforColorreactions Amino acid signified
Biuret peptide bond Peptidebond(Tripeptideonwards)
Ninhydrin Alpha-amino acids All Alpha-amino acids
Xanthoproteic Aromatic/benzene ring Phenylalanine,Tyrosine,Tryptophan
Million's Phenolic group Tyrosine
Sakaguchi Guanidino group Arginine
Lead acetate Sulfhydryl Cysteine
Hopkins Cole’s
aldehyde
Indole group Tryptophan
Nitroprusside Sulfhydryl Cysteine
Pauly’s Imidazole Histidine
FolinCocteau's Phenol group Tyrosine
119. Biuret Test Observation Inference
1. Biuret Test
2ml O.S. + 2ml 5% NaOH + 2 drops of
1% CuSO4 & Mix.
Control –
2ml D/W + 2ml 5% NaOH + 2 drops
of 1% CuSO4 & MIX
Violet color
Blue color
1. In alkaline medium, peptide bond
nitrogen in proteins/polypeptide
molecule react Cupric ions from
CuSO4 to form violet color
complex. A group test for
proteins.
2. All proteins & peptides
possessing at least two peptide
linkages i.e. with tri peptides
(with 3 amino acids and 2
peptide bonds ) give biuret test
positive . Histidine is only amino
acid that answers biuret test.
3. Testissonamedasitisgivenbythe
compoundBiuret-anonproteinorganic
compoundformedonheating urea.
2moleculesofureacondensetoform
Biuretat180 C .
Applications of Biuret Test : 1. As a Qualitative test for detection of proteins with minimum
two peptide bonds 2. Quantitative estimation of serum protein
Individual amino acids and
dipeptides do not answer this test.
Magnesium and ammonium
sulphates interfere with this
reaction.
120. Copper coordinated complex formed in Biuret Test
Biuret Test :is answered by compounds containing two or more CO-NH groups i.e. peptide
bonds . All proteins and peptides possessing at least two peptide linkage i.e. tripeptide (with
3 amino acids) give positive Biuret Test .
Cu 2+
C
II
O
NHHN
O
II
C
C
II
O
O
II
C NH
NH
NH
NH
Copper coordinated complex formed in Biuret Test
➢ Histidine is the only amino acid that answers Biuret Test.
NH2 two molecules of Urea
I
C= O NH2
I I
NH2 180 C C=O Biuret
+ I
NH2 NH
I I
C=O C=O
I I
NH2 NH2
122. Ninhydrin Test :Color reactions of proteins & amino acids
Principle: alpha amino acids react with Ninhydrin & by oxidative decarboxylation
forms CO2 + NH3 + Aldehyde. The reduced Ninhydrin reacts with liberated NH3 forming
purple color complex.( Rheumann’s purple)
Applications of Ninhydrin Test :
1. Quantitative estimation of amino acids
2. Used as a spray for detection of amino acids on paper chromatogram
123. Ninhydrin Test Observation Inference
2ml OS + 0.5 ml of 1%
Ninhydrin . Boil for 1 min.
and then Cool
Purple color Test answered by all -amino acid and
N-terminal amino group of proteins .
Principle: -amino acid react with 2
molecules of Ninhydrin & by oxidative
decarboxylation forms CO2 + NH3 +
Aldehyde with one carbon atoms less .
The reduced Ninhydrin reacts with
liberated NH3 forming purple color
complex.( Rheumann’s purple)
Applications of Ninhydrin Test :
1. Quantitative estimation of amino acids
2. Used as a spray for detection of amino acids on paper chromatogram.
Proline and hydroxy proline: give yellow color
Ninhydrin Test :Color reactions of proteins & amino acids
124. Ninhydrin reaction
COO
-
+ H3N +- C - H
R
+ 3 H2O + H +
Ninhydrin→ Ninhydrin
-Amino acid
Heat H
CO2 + R- C
O
C= N-C H
O
II
C
O
II
C
O
II
C
C
II
O
C
II
O
C
II
O
C
II
O
I
I
OH
C
OH
OH
C
OH
Purple
pigment
O
II
C
126. Xanthoproteic Test observation inference
2ml OS + 2ml concentrated
HNO3 .Boil, cool & add 40%
NaOH drop by drop
White precipitate turning
yellow upon heating &
orange with 40% NaOH
Principle: Concentrated HNO3
forms a white ppt of protein
turning yellow upon heating
due to formation of nitro –
compounds of aromatic
amino acids Tyrosine,
Tryptophan & Phenyl alanine.
On addition of alkali sodium
salts of the nitro compound
is formed . Given positive by
Tyrosine having phenolic
group with OH .
Application of Xanthoproteic Test : given by benzene ring containing amino acids i .e.
Tyrosine ,Tryptophan & Phenylalanine
Xanthoproteic Test :Color reactions of proteins & amino acids
This reaction causes the yellow stain
In skin by nitric acid.
127. Xanthoproteic and Millon’s Test for detection of aromatic amino acids
Observation : Brick red color precipitate and pink solution
129. Hopkin’s Test for detection of Tryptophan
Tryptophan containing protein is mixed with glyoxylic acid and the mixture is
layered with concentrated sulfuric acid . A violet ring at the interfere of liquids
show the presence of the indole ring .
130. Hopkin’s Test for detection of Tryptophan
Tryptophan containing protein is
mixed with glyoxylic acid and the
mixture is layered with
concentrated sulfuric acid . A
violet ring at the junction of
liquids infer presence of the
indole ring of Tryptophan .
131. Cole’s aldehyde test observation Inference
2ml of OS + 1 drop of
Mercury sulphate (10% )
+ 1 drop of 1:800 formalin
solution + 2ml of H2SO4 to be
added from side of Test Tube.
Do not mix .
Violet ring at the junction of
two layers
Given by Tryptophan having
indole group .
Formaldehyde & indole
group of Tryptophan
condense in presence of
H2SO4.
The condensation product
forms a purple color complex
with mercury from mercury
sulphate.
Hopkin Cole’s aldehyde test for detection of Tryptophan
Application of Cole’s aldehyde test : Given by Tryptophan having indole group .
Urinary examination for Hartnup’ s disease →Test positive
132. Hopkin Cole’s aldehyde test for detection of Tryptophan
Observation:
Violet ring at the junction of two
layers.
Gelatin with limited Tryptophan
content will not answer this test.
Hopkin Cole’s aldehyde test :given by
Tryptophan having indole group .
Formaldehyde & indole group of
Tryptophan condense in presence of
H2SO4. The condensation product forms
a purple color complex with mercury
from mercury sulphate.
133. Modified Million's Test Observation Inference
2ml OS + 2 ml Mercuric
sulphate (10% ) in H2SO4 Boil
continuously for 1 minute
then cool ,Add 1ml of freshly
prepared 1 % NaNO2 solution.
Heat observe color of
precipitate or solution.
Brick red color precipitate
and pink solution.
Principle : proteins are
precipitated by strong acids.
The phenolic group of
Tyrosine react with Mercury
sulphate in H2SO4 to form
Mercury –phenolate
(Tyrosine) complex. Red color
precipitate is due to nitration
by NaNO2 . Given by phenol
group containing amino acid .
Modified Millon's Test for detection of Tyrosine in urine :
color reactions of proteins & amino acids
Application of Modified Million's Test for detection of Tyrosine in urine ( Diagnostic test
for Tyrosinemia )
134. Modified Million's Test for detection of Tyrosine in urine
Observation : Brick red color precipitate and pink solution.
Proteins are precipitated by strong acids. The phenolic group of Tyrosine
react with Mercury sulphate in H2SO4 to form Mercury –phenolate
(Tyrosine) complex. Red color precipitate is due to nitration by NaNO2 .
Given by phenol group containing amino acid .
135. Sakaguchi Test Observation Inference
Sakaguchi Test :
2ml of OS + 1 drop of Alpha
Naphthol + 1ml 5 % NaOH +
10-12 drops freshly prepared
sodium hypobromite solution.
then Mix.
Red color Guanidine group of Arginine
forms a red colored complex
with Alpha-naphthol & Na
hypobromite in alkaline
medium.
Application of Sakaguchi Test : diagnosis of Hyperargininemia( inborn error in Arginine
metabolism due to defect in enzyme Arginase ).
SakaguchiTestfordetectionofArginine:colorreactionsofproteins&aminoacids
136. Sakaguchi Test for detection of Arginine
Guanidine group of
Arginine forms a red
colored complex
with Alpha-naphthol & Na-
hypobromite in alkaline
medium.
Application of Sakaguchi Test : diagnosis of Hyperargininemia
( inborn error in Arginine metabolism due to defect in enzyme Arginase ).
137. Lead acetate Test Observation Inference
2ml of OS + 2ml 40%
NaOH Boil for 2-3mins.
Cool and then add few
drops of lead acetate.
Black color Given positive by Cysteine & cystine
(after reduction) CH2-SH . Not given by
Methionine ( -CH2-SCH2 ) as the ‘S’ split
of as Na2S ( sodium sulfide) when the
protein is boiled with strong alkali. On
addition of lead acetate , black
precipitate of PbS (lead sulphide ) is
formed.
Application of Lead acetate Test: Test for sulphur containing amino acids
Cysteine & Cystine.
Albumin and keratin will answer sulphur test positive.
Casein will give this test negative.
Lead acetate Test : Color reactions of proteins & amino acids
138. Lead acetate Test : Color reactions of proteins & amino acids
Black precipitate of PbS
Applications of Lead acetate Test: Test for Sulphur containing amino acids Cysteine &
Cystine. Methionine does not answer Lead acetate Test as Sulphur in Methionine is in
the thioester linkage and not free for reaction with lead acetate . Albumin and keratin
will answer this sulphur test positive. Casein will give this test negative.
139. Nitroprusside reaction for SH groups
Protein with free sulfhydryl groups + Sodium nitroprusside +Ammonia→
reddish color
Denatured protein : positive test
Protein in native state : negative test
140. Test Observation Inference
Molisch's Test:
2ml OS + 2 drops alpha-
Naphthol . Then Mix .Add at
least 2ml of concentrated
H2SO4 from side of test tube
which is held tiled at angle of
45C.
Control:
2mlwater+2dropsof1%alpha-
Naphthol→Mix.Addatleast2ml
ofconcentratedH2SO4 fromsideof
testtubewhichisheldtiledatangle
of45C.
Purple color
ring
No Purple
color ring
Given by proteins since egg white solution
contains ovomucoid (a glycoprotein), the
carbohydrate gives a positive reaction.
Applications of Molisch's Test: used for detection of presence of Glycoproteins in
conjugation with positive Biuret Test.
Molisch's Test for detection of Glycoproteins
141. Molisch's Test for detection of Glycoproteins
Molisch'sTest isgivenbyproteinssinceeggwhitesolutioncontainsovomucoid(aglycoprotein),the
carbohydrategivesapositivereaction.
ApplicationsofMolisch'sTest:usedfordetectionofpresenceofGlycoproteinsinconjugationwithpositive
BiuretTest
142. Test Observation Inference
Test for Histidine : 3ml OS +
10 drops H2SO4 + 10 drops
freshly prepared NaNO2 .Wait
for 3mins .Add 8 drops of
Na2CO3 .
Diazo –benzene –sulfonic
acid reagent
Cherry Red color
solution
Test is given positive by Imidazole ring of
Histidine to give diazotized product under
alkaline condition .
Same reagent will give an orange red
colored product with phenol group of
Tyrosine.
Pauly’s Test for Histidine :Color reactions of proteins & amino acids
143. Clinical applications of Color Reactions of Proteins & Amino Acids
❖Color reactions are due to specific functional groups present in
amino acids and proteins .These reactions are important in the
following ways.
❖Clinical applications of Color Reactions of Proteins & Amino Acids:
• Used in qualitative identification & quantitative estimation of amino
acids or peptides or proteins containing these amino acids .
• Used in spray for detection of specific amino acids on paper
chromatograms .
144. Chromatography for amino acid determination :
comparison of RF value with standard amino acids
Rf value of each amino acids is its characteristic property. Thus ,the amino acids in the
unknown mixture can be identified fairly well by comparing their Rf values with those of pure
amino acids.
145. Identification of amino acids is done using chromatography
Rf value of each amino acids is its characteristic property. Thus ,the amino acids in the
unknown mixture can be identified fairly well by comparing their Rf values with those of
pure amino acids.
147. Quantitative estimation of proteins
❖Techniques used for Quantitative estimation of proteins :
1. Kjeldahl procedure
2. Biuret method
3. Lowry method
4. Spectrophotometric estimations
5. Radial immunodiffusion ( Mancini technique)
6. Nephelometry
7. Turbidometry
8. RIA and ELISA tests
148. Kjeldahl procedure : Quantitative estimation of proteins
1. Estimation of plasma protein concentration is based on chemical
composition:
A. Peptide bonds:
i. Biuret method :–peptide bond of proteins +copper (Cu2+) + alkaline pH
→violet color complex
ii. Ultra- violet absorption by peptide bond :Spectrophotometer
Micro gram protein = (optical density 215 - optical density 225)x144
B. Nitrogen content :weight contribution in protein by nitrogen 16%(kjeldahl
method for N content ) =weight of protein =N X 6.25
149. Kjeldahl procedure for Quantitative estimation of proteins
• The protein sample is digested by boiling (160 C) with concentrated
Sulphuric acid in presence of copper sulphate and sodium sulphate as
catalyst.
• The nitrogen content is reduced to ammonia which is absorbed by acid
medium to become ammonium sulphate
• Cooling followed by the digest is made alkaline by addition of excess alkali
• Ammonia is liberated, which is absorbed by known quantity of standard
acid kept in a vessel.
• The excess acid present in the vessel is back titrated with a standard base ,
from which the liberated ammonia is calculated .
• The quantity of nitrogen present in protein is assessed. On average nitrogen
of protein is 16%.
• Weight of protein in original sample =Nitrogen content X 6.25
150. Kjeldahl procedure for Quantitative estimation of proteins
The protein sample is digested by boiling (160 C) with concentrated Sulphuric acid in presence of copper
sulphate and sodium /potassium sulphate as a catalyst. The nitrogen content is reduced to ammonia which is
absorbed by acid medium to become ammonium sulphate . Cooling followed by the digest is made alkaline by
addition of excess alkali. Ammonia is liberated, which is absorbed by known quantity of standard acid kept in a
vessel. The excess acid present in the vessel is back titrated with a standard base , from which the liberated
ammonia is calculated .
151. Biuret method :Quantitative estimation of proteins
❖Principle of Biuret method for Quantitative estimation of proteins :
Cupric ions chelate with peptide bonds of proteins in alkaline medium to
produce pink or violet color . The intensity of violet color is proportional to the
number of peptide bonds. The intensity of color of test and standard is
measured colorimetrically . The intensity of color of test is compared with
that of a standard protein solution treated with this reagent similarly.
Concentration of protein = OD test /OD std x concentration of std ( Beer’s
Lambert law)
152. Copper coordinated complex formed in Biuret Test
Biuret Test :is answered by compounds containing two or more CO-NH groups i.e. peptide
bonds . All proteins and peptides possessing at least two peptide linkage i.e. tripeptide (with
3 amino acids) give positive Biuret Test .
Cu 2+
C
II
O
NHHN
O
II
C
C
II
O
O
II
C NH
NH
NH
NH
Copper coordinated complex formed in Biuret Test
➢ Histidine is the only amino acid that answers Biuret Test.
NH2 two molecules of Urea
I
C= O NH2
I I
NH2 180 C C=O Biuret
+ I
NH2 NH
I I
C=O C=O
I I
NH2 NH2
154. Lowry method: Quantitative estimation of proteins
❖Principle of Lowry method for Quantitative estimation of proteins:
It is based on the reduction of Folin-Ciocalteau phenol reagent (phospho
tungstic acid and phosphomolybdic acid) by Tyrosine and Tryptophan
residues of protein. A blue color is developed . The intensity of color of test
and standard is measured colorimetrically . The intensity of color of test is
compared with that of a standard protein solution treated with this reagent
similarly.
❖Concentration of protein = OD test /OD std x concentration of std ( Beer’s
Lambert law)
155. Spectrophotometric estimations for Quantitative estimation
of proteins
❖Principle of Spectrophotometric estimations for Quantitative estimation of
proteins : proteins will absorb ultraviolet light at 280 nm . This is due
Tyrosine and Tryptophan residues in the protein solution. Quantitation is
done by comparing the absorbance of the test solution with a known
standard .
Ultra- violet absorption by Tyrosine and
Tryptophan residues
156. Radial immunodiffusion (Mancinitechnique)forQuantitativeestimationofproteins
• Radial Radio
• Radial immunodiffusion : the precipitation of arc is moving radially outward from
the point of application ( the name radial) .
• The specific antibody is incorporated in the liquid agar then allowed to solidified
on a glass.
• The small wells (1 mm diameter) are cut in the agar and antigen (protein solution
or patient’s serum) is added in the well.
• The plate is incubated at 4 C for 1 to 3 days .
• The antigen molecules diffuse radially around the wells and react with the specific
antibody molecules present all over the agar.
• A white ring of precipitation is seen where equimolar concentration ( 1:1 ratio) of
antigen and antibody is attained.
• The diameter of the precipitation ring will be proportional to the log of antigen
concentration
• If known standards of different concentration is included along with test, standard
graph is plotted. The quantity of test substance can be obtained by plotting a
perpendicular on X –axis.
157. Radial immunodiffusion
Radial immunodiffusion Antigen on log scale
If known standards of different concentration is included
along with test, standard graph is plotted. The quantity of
test substance can be obtained by plotting a perpendicular
on X –axis.
158. Nephelometry for Quantitative estimation of proteins
❖Principle of Nephelometry: is based on the measurement of scattering of
light by colloids and defined as the quantitation of light scattered by turbid
particles ( antigen –antibody complex ) in solutions.
❖ Scattering of light by turbid particles (antigen –antibody complex) in
solutions was studied originally by Richard Zsigmondy (Noble 1925) and
used as quantitation method by Libby in 1938.
159. Estimation of albumin by Nephelometry
Specific antibody against albumin is added to the solution.
The resultant antigen –antibody is formed and it imparts turbidity to the solution.
A beam of light (preferably laser beam) is passed through solution.
Particles in the solution scatter the light.
Thelightturningat30to90 (generally60)iscollectedandpassedintoadetectorsystem.
Theemergentscatteredlightwillbeproportionaltothe turbidityofthesolution,whichinturnwillbe
proportionaltotheantigen.
Quantitation of albumin is done using the standard of known concentration.
160. Turbidometry for Quantitative estimation of proteins
❖Principle of Turbidometry for Quantitative estimation of proteins :
proteins in biological solutions like urine and cerebrospinal fluid(CSF) can be
estimated by adding protein precipitating agents ( sulfosalicylic acid or
antibody) . The turbidity thus produced is measured. Quantitation of protein
is done using the standard of known concentration.
161. Comparison of Turbidometry and Nephelometry
TurbidometryandNephelometryarebasedonthesameprincipleofscatteringoflightbycolloidalparticles.
In Nephelometry , the emergent light by scatter light at 60 is observed .
In Turbidometry , the emergent light by scatter light at 180 is observed .
163. Flocculation tests as Liver Function Tests based upon Protein Metabolism
Principle : The stability of protein in solution (serum ) in presence of precipitating
agents depend upon A/ G ratio .
1. Thymol turbidity test
Thymol decreases solubility of lipids & dispersion of beta & Gamma globulin
Saturated thymol solution + serum → 30 mins turbidity of test solution is compared
with standard solution of concentration 10mg/100ml
slight turbidity ( 0-4 units ) : observed in normal /obstructive jaundice
Increased turbidity – increased in globulins → observed in infective hepatitis
2. Serum colloidal gold test
3. Serum colloidal test
4. Zinc sulphate test
Positive flocculation test suggest increase in gamma globulin & lipoprotein observed in
liver diseases ,Kala azar ,Multiple myeloma
164. Principle of Enzyme Linked Immunosorbent Assay—(ELISA )
Principle of Enzyme Linked Immunosorbent Assay (ELISA ) is as
follows :
• ELISA is based on the immunochemical principle of antigen antibody
reactions .
• It is based on the specificity of antigen-antibody complex formation
and its detection by a second antibody conjugated with suitable
enzyme such as peroxidase.
165. Principle of Enzyme Linked Immunosorbent Assay(ELISA)
ELISA is based on the immunochemical principle of antigen antibody reactions . It is based on
the specificity of antigen-antibody complex formation and its detection by a second antibody
conjugated with suitable enzyme such as peroxidase.
168. Principle of Radioimmunoassay(RIA)
❖Principle of Radioimmunoassay(RIA) :combine the principles of radioactivity of isotopes and
immunological reactions of antigen and antibody.
• It is based on the competition between the labelled and unlabeled antigens to bind with
antibody to form antigen –antibody complexes (either the labelled and unlabeled) .
• The unlabeled antigen ( e.g. insulin) is the substance to be assayed .
• The specific antibody (Ab) is then subjected to react with unlabeled antigen in the presence of
excess amounts of isotopically labelled (131I) antigen (Ag +) with known radioactivity.
• There occurs a competition between the antigens labelled and unlabeled (Ag and Ag +) to bind
the antibody to form antigen –antibody complexes (either the labelled and unlabeled).
• As the concentration of unlabeled antigen (Ag) increases ,the amount of labelled antigen –
antibody (Ag + Ab) decreases .
• Thus, the concentration of labelled antigen –antibody(Ag + Ab) is inversely related to the
concentration of unlabeled antigen (Ag) i.e. substance to be assayed .
• This relation is almost linear on log scale . A standard curve can be dawn by using different
concentrations of unlabeled antigen (Ag) along reacted with the same quantities of antibody
and labelled antigen.
• labelled antigen –antibody(Ag + Ab) is separated by precipitation . The radioactivity of isotope
(131I) present in labelled antigen –antibody(Ag + Ab) is measured.
169. The specific antibody (Ab) is subjected to react with unlabeled antigen in the
presence of excess amounts of isotopically labelled (131I) antigen (Ag +) with
known radioactivity. There occurs a competition the antigens labelled and
unlabeled (Ag and Ag +) to bind the antibody to form antigen –antibody
complexes (either the labelled and unlabeled).
Competition between the labelled and unlabeled antigens to bind with
antibody in reaction of Radioimmunoassay(RIA)
170. The concentration of labelled antigen –antibody(Ag + Ab) is inversely related to the
concentration of unlabeled antigen (Ag) i.e. substance to be assayed .
This relation is almost linear on log scale . A standard curve can be dawn by using different
concentrations of unlabeled antigen (Ag) along reacted with the same quantities of antibody
and labelled antigen.
A standard curve of Radioimmunoassay(RIA)
171. RIA and ELISA tests for Quantitative estimation of proteins
172. Advantages and Disadvantages of Techniques used for Quantitative estimation of proteins:1
Techniques Advantages Disadvantages
Kjeldahl procedure Most accurate , precise ,easier method, used
for standardizing a particular protein
Takes many days to get result and
unsuitable for routine clinical
work
Biuret method Simple and one step process ,widely used
method for plasma protein
less sensitivity, unsuitable for
estimation of protein in mg/
microgram quantity
Lowry method Very sensitive, suitable for estimation of protein
in mg/ microgram quantity
If Tyrosine and Tryptophan
content of test protein and
standard vary widely, then
accuracy is lost
Spectrophotometric
estimations
Accurate , simple and highly sensitive up to
microgram quantities, protein is not wasted in
this method since color reaction is not
employed
Instrument required is costly
Nephelometry rapid method ,suitable for automated program Needs careful standardization ,
reagents and Instruments
required costly
173. Advantages of Techniques used for Quantitative estimation of proteins:2
Techniques Advantages
Radial immunodiffusion ( Mancini
technique)
Simple and sensitive to quantitate mg/
microgram quantities of protein, routine
method for estimation of
immunoglobulins and complement
component as it is specific because it
employs antibodies
Turbidometry Specific , simple ,easy to perform and
cheap
Radioimmunoassay( RIA )and
Enzyme linked immunosorbent assay
(ELISA) tests
sensitive to quantitate nanogram and
picogram quantities of protein
174. Albuminuria
❖Conditions associated with Albuminuria:
1. Nephrotic syndrome
2. Inflammation of urinary tract
3. Microalbuminuria: (30-300mg/day)→ prediction of risk of renal
disease
175. Biochemical test to detect Proteinuria
❖Conditions associated with Albuminuria:
1. Nephrotic syndrome
2. Inflammation of urinary tract
3. Microalbuminuria: (30-300mg/day)→ prediction of risk of renal disease
176. Biochemical Test for Proteinuria
❖Biochemical Test for Proteinuria : The Proteinuria is commonly assessed
by the heat test and Acetic acid test.
❖Overnight first voided urine sample ( early morning urine EMU) may be
used for measurement of protein as it rules out the possibility orthostatic
albuminuria.
1.Acetic acid test : Urine + 1% Acetic acid dropwise (to achieve isoelectric
protein of albumin )→ boil → cloudiness → proteinuria
Calcium & Magnesium phosphate precipitate on heating ( precipitate
soluble in acidic medium )
2.Elisa
3. Radial micro diffusion (detection of microalbumin )
4.Immunoturbidometry
5. Dipstick –replacing the old method
177. Serum proteins and their functions
Serum proteins Normal level g/ dl % of total protein Functions
Albumin 3.2- 5.1 56 Stabilizer of blood volume , transporter of
fatty acids and bilirubin, regulator of fluid
exchange between the vascular
compartments by exerting colloid pressure
1- Globulin 0.06-.39 5 Consist of vitamin A binding protein (retinol)
2- Globulin 0.28-0.74 9 Consist of ceruloplasmin which is copper
containing protein ,involved in iron
absorption
-Globulin 0.69-1.25 11 Consist of transferrin – an iron transport
protein
-Globulin 0.8 -2.0 19 Called as immunoglobulins confer immunity
against infectious diseases
Fibrinogen 0.2-0.4 Forms clots during coagulation process
Total 6.3-7.8 7g/dl Most proteins are synthesized in liver ,
immunoglobulins are synthesized in lympho –
reticular system .
Albumin: Globulin ratio = 1.5 : 2
179. Abnormal serum protein electrophoretic patterns in liver diseases
Liver disease Abnormal serum protein electrophoretic pattern
Acute hepatitis Pre albumin reduced
Cirrhosis Albumin reduced, gamma globulins increased
Hepatocellular disease Alpha- 1 globulins reduced (parallel to albumin)
Biliary obstruction Alpha- 2 globulins ,beta globulins increased
➢ The rise in gamma globulins will have wide base ,suggestive of polyclonal
gammopathy .
180. Protein concentration in cerebrospinal fluid in health and diseases
❖Normal/physiologicalProteinconcentrationincerebrospinalfluid(CSF):15-45mg/dl
❖Increased Protein concentration in cerebrospinal fluid observed in :
1. Tubercular meningitis
2. Bacterial meningitis
3. Brain tumor
4. Subarachnoid hemorrhage
