This presentation shows about how the determination of molecular weight is done and what are the different ways or method to determine the molecular weight. This presentation also tells about the enzymes and its characterization.
3. ENZYMES
• Basic Definition :
Enzymes are catalysts that increase the rate of a chemical reaction
without being changed themselves in the process.
Enzyme activity can be regulated, varying in response to the
concentration of substrates or other molecules. They function under strict
conditions of temperature and pH in the body.
Enzymes are proteins that act as biological catalysts. Catalysts accelerate
chemical reactions. The molecules upon which enzymes may act are called
substrates, and the enzyme converts the substrates into different
molecules known as products
4. SOME BASIC PROPERTIES OF
ENZYMES
• Catalytic Property.
• Specificity.
• Reversibility.
• Sensitiveness to Heat and Temperature.
• Specific to Hydrogen Ion Concentration (pH)
5. MOLECULAR WEIGHT
DETERMINATION OF ENZYMES
• When an enzyme has been purified, its molecular weight, more correctly
termed relative molecular mass, Mr, may be determined.
• It can be done by 3 ways:-
a) Gel electrophoresis (SDS-PAGE)
b) Chromatography (size exclusion chromatography and gel filtration
chromatography)
c) Spectroscopy (Mass Spectroscopy)
6. GEL ELECTROPHRESIS (SDS-PAGE)
• Most popular method of determining Mr of an enzyme after purification
is PAGE (Poly Acrylamide Gel Electrophoresis) , in the presence of SDS
(Sodium Dodecyl Sulphate) & a reducing agent (2-mercaptoethanol).
SDS complexes with protein and denatures the protein as well as
imparts a negative charge to it.
• Basic Principle
• SDS-PAGE is an electrophoresis method that allows protein separation
by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-
based discontinuous gel. In addition, SDS (sodium dodecyl sulfate) is
used. About 1.4 grams of SDS bind to a gram of protein, corresponding
to one SDS molecule per two amino acids.
7. • Why SDS PAGE used for
proteins?
• For proteins, Sodium Dodecyl Sulfate (SDS) is
used to linearize proteins and to negatively charge
charge the proteins. ... As a result, negatively
charged proteins will migrate towards the positive
electrode and will be fractionated by approximate
size during electrophoresis. This procedure is
called SDS-PAGE.
• How does SDS PAGE gel work?
• SDS-PAGE separates proteins primarily by
mass because the ionic detergent SDS
denatures and binds to proteins to make them
uniformly negatively charged. Thus, when a
current is applied, all SDS-bound proteins in a
sample will migrate through the gel toward
8. CHROMATOGRAPHY
• Chromatography is a physical method of separation that distributes
components to separate between two phases, one stationary
phase), the other (the mobile phase) moving in a definite direction.
eluate is the mobile phase leaving the column. ... The eluent is the
solvent that carries the analyte.
• Size exclusion chromatography
• Size-exclusion chromatography (SEC), also known as molecular sieve
chromatography, is a chromatographic method in which molecules in
solution are separated by their size, and in some cases molecular
weight. It is usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers.
9. • Basic Principle
• The number average molecular weight (Mn) is
calculated by dividing the total polymer weight by the
total number of polymer molecules, using equation (1).
The weight average molecular weight (Mw) is
calculated using equation (2), which emphasizes the
contribution of polymers with larger molecular
weights.
• How does size exclusion
chromatography work?
• Size exclusion chromatography (SEC) separates
molecules based on their size by filtration through a
gel. ... Small molecules diffuse into the pores and their
flow through the column is retarded according to their
size, while large molecules do not enter the pores and
are eluted in the column's void volume.
10. •Gel Filtration chromatography
• Gel filtration is a technique in which the separation of components is
based on the difference in molecular weight or size. It is the simplest
and mildest of all the chromatography techniques and separates
molecules on the basis of differences in size.
• Principle.
• The gel filtration chromatography is based on the molecular size and
the hydrodynamic volume of the components. The molecules are
separated by the differential exclusion or inclusion of solutes as they
pass through the stationary phase containing heteroporous cross-
polymeric gel or beads.
• Gel filtration chromatography is an established method for
determining the size and molecular mass of proteins. Fractionation is
based on the diffusion of molecules into the pores of the resin.
11. • How does gel filtration chromatography work?
• In a gel filtration chromatography column, the stationary phase is
composed of a porous matrix, and the mobile phase is the buffer that
flows in between the matrix beads. Molecules and complexes that are
too large to enter the pores stay in the mobile phase and move through
the column with the flow of the buffer.
12. MASS SPECTROSCOPY
• Mass spectrometry (MS) is an analytical technique that measures the
mass-to-charge ratio of ions. The results are typically presented as a
mass spectrum, a plot of intensity as a function of the mass-to-charge
ratio.
• Basic Principle
• A mass spectrometer generates multiple ions from the sample under
investigation, it then separates them according to their specific mass-
charge ratio (m/z), and then records the relative abundance of each
type.
• What is mass spectroscopy used for?
• Mass spectrometry is an analytical tool useful for measuring the mass-
to-charge ratio (m/z) of one or more molecules present in a sample.
These measurements can often be used to calculate the exact
13. how does it work?
• A mass spectrometer produces charged particles (ions) from the chemical substances
that are to be analyzed. The mass spectrometer then uses electric and magnetic fields
to measure the mass ("weight") of the charged particles.
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14. WHAT ARE THE MAIN CHARACTERISTICS
OF AN ENZYME?
• Speed up chemical reactions.
• They are required in minute amounts.
• They are highly specific in their action.
• They are affected by temperature.
• They are affected by pH.
• Some catalyze reversible reactions.
• Some require coenzymes.
• They are inhibited by inhibitors.
15. • Enzymes speed:- Enzymes are usually helps in increasing the speed of catalysis
process. Enzymes speed the rate of a reaction by lowering the amount of activation
energy required to reach the transition state, which is always the most difficult step in
a reaction.
• Specificity:- Most of the enzymes are very specific in nature. They act on the their
particular related substrate. Specificity is the ability of an enzyme to choose exact
substrate from a group of similar chemical molecules. The specificity is actually a
molecular recognition mechanism and it operates through the structural and
conformational complementarity between enzyme and substrate.
• Temperature:- Enzyme activity increases as temperature increases, and in turn
increases the rate of the reaction. This also means activity decreases at colder
temperatures. All enzymes have a range of temperatures when they are active, but
there are certain temperatures where they work optimally.
16. • pH :- Enzymes are affected by changes in pH. The most favorable pH value - the
point where the enzyme is most active - is known as the optimum pH. Extremely high
or low pH values generally result in complete loss of activity for most enzymes.
• Coenzyme :- Coenzymes are small molecules. They cannot by themselves
catalyze a reaction but they can help enzymes to do so. In technical terms,
are organic nonprotein molecules that bind with the protein molecule (apoenzyme)
form the active enzyme (holoenzyme). Unlike the inorganic cofactors, coenzymes are
organic molecules. Certain enzymes need coenzymes to bind to the substrate and
cause a reaction. Since the coenzymes are changed by the chemical reaction, these
considered to be secondary substrates of the reaction.
• Inhibitors :- Enzyme inhibitors are substances which bind to the enzyme with
resulting loss of activity, without damaging the enzyme's protein structure. These
substances which alter the catalytic action of the enzyme and consequently slow
down, or in some cases, stop catalysis.
