!Experiment 1
Transcript of !Experiment 1
Experiment 1
Department of Biological Pharmacy, China Pharmaceutical University, 2011
Preparation of Elastic Protease from Pancreas and Analysis of Enzyme Activity
CONTENTS
Part I: Preparation of Elastic Protease2
Part II: Analysis of Enzyme Activity3
Purposes1
Purposes
Learn the principle of preparation of elastic protease from pancreas.
Master the analysis method of enzyme activity.
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Part I
Preparation ofElastic Protease
Procedures I
Frozen Pancreas75 g
Frozen Pancreas75 g
Pancreatic Plasma
Pancreatic Plasma
(1) Activate the enzyme at 24 for 24h℃(2) remove the fat, cut into pieces(3) add 50ml HAc-buff ,mashed
FiltrateFiltrate
(1) add 250 mL HAc-buff, adjust pH to 4.5 (2) 25℃ stir for 1 hours, (3) Filter
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Procedures II
Continued from previous page …Continued from previous page …FiltrateFiltrate
Resin(to absorb protein)
Resin(to absorb protein)
(1) add 40 g resin for adsorption(2) 25℃ stir for 1.5 h (3) wash with water
EluateEluate
(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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Procedures III
Continued from previous page …Continued from previous page …EluateEluate
PrecipitationPrecipitation
Add 3-time-volumn cold acetone
Elastase powderElastase powder
(1) wash twice with acetone(2) wash once with ether(3) vacuum drying
End Product Obtained
Part II
Analysis ofEnzyme Activity
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.
Preparation of Reference Substance I
30 mg Congo red elastin
(taken accurately)
1 220 mL standard elastic enzyme
solution(must be sufficient)
100 mLvolumetric flask
3Keep the flask at 37 in a ℃ water
incubator
4Shake the bottle gently
until all substrates dissolves ( about 60 min)
Preparation of Reference Substance II
6add boric acid buff
to the scale
3Keep the flask at 37 in a ℃ water
incubator
4Shake the bottle gently
until all substrates dissolves ( about 60 min)
50 mL phosphate buffer (pH 6.0)
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100 mLvolumetric flask
Sample Preparation
5 mg enzyme sample
(taken accurately)
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mortar
25 mL pH 8.8 boric
acid buff(add a small
amount at first)
43grind until everything
dessolves Dilute enzyme solution with boric acid buff to 2 ~ 3 unit/mL
1 mL boric acid buff
Standard CurveTake 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
Mixture of buffer (mL) 10.0 8.0 6.0 4.0 2.0 0
A495 value
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Sample Preparation
Take 3 tubes to operate according to the table below:
Tube No. 0 1 2
Congo red elastin (mg) 1.2 3 3
pH 8.8 boric acid buff (mL) 5 4 4
Await measuring enzyme liquid (mL)
- 1 1
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
CaculationTake the average absorption and check the corresponding enzyme activity according to the standard curve.
Calculate the relative enzyme activity (enzyme activity/enzyme amount) by taking into account the dilution times.
Calculate the total enzyme recovery yield.
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The End
It’s time for your practice. EVERYBODY GO GO GO!
PPT &制作:设计
中国 科大学药 · 生物制 教研室 药 2011.9