!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.

1

2

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

Continued on next page …

Continued on next page …

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

Continued on next page …

Continued on next page …

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)

5

100 mLvolumetric flask

Sample Preparation

5 mg enzyme sample

(taken accurately)

1

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

12

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