1. E
xperiment 1
Preparation of Elastic Protease from Pancreas
and Analysis of Enzyme Activity
Department of Biological Pharmacy, China Pharmaceutical University, 2011

2. CONTENTS
1
Purposes
2
Part I: Preparation of
Elastic Protease
3
Part II: Analysis of
Enzyme Activity

3. Purposes
1
Learn the principle of preparation
of elastic protease from pancreas.
2
Master the analysis method of
enzyme activity.

4. Part I
Preparation of
Elastic Protease

5. Procedures I
Frozen Pancreas
Frozen Pancreas
75 g
75 g
(1) Activate the enzyme at 24℃ for 24h
(2) remove the fat, cut into pieces
(3) add 50ml HAc-buff ,mashed
Pancreatic
Pancreatic
Plasma
Plasma
(1) add 250 mL HAc-buff, adjust pH to 4.5
(2) 25℃ stir for 1 hours,
(3) Filter
Continued on
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Filtrate
Filtrate
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6. Procedures II
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Filtrate
Filtrate
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(1) add 40 g resin for adsorption
(2) 25℃ stir for 1.5 h (3) wash with water
Resin
Resin
(to absorb protein)
(to absorb protein)
(1) add 50 ml 1 M NH4Cl-buff, adjust pH to 9.0
(2) stir for 1 h (3) adjust pH to 5.2~6.0
(4) filter
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Continued on
Eluate
Eluate
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7. Procedures III
Continued from
Continued from
Eluate
Eluate
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Add 3-time-volumn cold acetone
Precipitation
Precipitation
(1) wash twice with acetone
(2) wash once with ether
(3) vacuum drying
Elastase powder
Elastase powder
End Product Obtained

8. Part II
Analysis of
Enzyme Activity

9. Definition
Definition of Enzyme Activity
The amount of enzyme hydrolyzing 1.0 mg Congo
red elastin within 20 min at pH 8.8 and 37 ℃ is
defined as an activity unit.

10. Preparation of
Reference Substance I
1
30 mg Congo red
elastin
(taken accurately)
4
Shake the bottle gently
100 mL
until all substrates
volumetric flask
dissolves ( about 60 min)
2
20 mL standard
elastic enzyme
solution
(must be sufficient)
3
Keep the flask at
37 ℃ in a water
incubator

11. Preparation of
Reference Substance II
5
50 mL phosphate
buffer (pH 6.0)
4
Shake the bottle gently
100 mL
until all substrates
volumetric flask
dissolves ( about 60 min)
6
add boric acid buff
to the scale
3
Keep the flask at
37 ℃ in a water
incubator

12. Sample
Preparation
1
2
5 mL pH 8.8 boric
acid buff
(add a small
amount at first)
5 mg enzyme
sample
(taken accurately)
mortar
3
grind until everything
dessolves
1 mL
boric acid buff
4
Dilute enzyme solution with boric
acid buff to 2 ~ 3 unit/mL

13. Standard Curve
1
2
Take series amounts of reference substance solution,
adding mixture of boric acid buffer and phosphate acid
buffer (1:1), as indicated in the table below.
Measure the absorption at 495nm, using the absorption
of tube 0 as control. Plot a standard curve.
Tube No
0
1
2
3
4
5
Reference substance (mL)
0
2.0
4.0
6.0
8.0
10.0
10.0
8.0
6.0
4.0
2.0
0
Mixture of buffer (mL)
A495 value

14. Sample
Preparation
Take 3 tubes to operate according to the table below:
Tube No.
Congo red elastin (mg)
pH 8.8 boric acid buff (mL)
0
1
2
1.2
3
3
5
4
4
Await measuring enzyme liquid
1
1
(mL)
Hydrolyze the mixure at 37℃ for 20 min (The intermittence is
stirred 20 or more times)
pH 6.6 phosphoric acid buff (mL)
5
5
5
Take the supernatant after centrifugation (3000 rpm × 10 min)
A495 value

15. Caculation
1
Take the average absorption and check the
corresponding enzyme activity according to
the standard curve.
2
Calculate the relative enzyme activity (enzyme
activity/enzyme amount) by taking into
account the dilution times.
3
Calculate the total enzyme recovery yield.

16. The End
It’s time for your practice.
EVERYBODY GO GO GO!
PPT 设计＆制作：
中国药科大学 · 生物制药教研室 2011.9