胰弹性蛋白酶的制备与分析.pptVIP

Experiment 1 Department of Biological Pharmacy, China Pharmaceutical University, 2011 CONTENTS Purposes Learn the principle of preparation of elastic protease from pancreas. Master the analysis method of enzyme activity. Part I Preparation ofElastic Protease Procedures I Procedures II Procedures III Part II Analysis ofEnzyme Activity Definition Preparation of Reference Substance I Preparation of Reference Substance II Sample Preparation Standard Curve Sample Preparation Caculation Take 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. The End * * * Preparation of Elastic Protease from Pancreas and Analysis of Enzyme Activity Part I: Preparation of Elastic Protease 2 Part II: Analysis of Enzyme Activity 3 Purposes 1 1 2 Frozen Pancreas 75 g Pancreatic Plasma (1) Activate the enzyme at 24℃ for 24h (2) remove the fat, cut into pieces (3) add 50ml HAc-buff ,mashed Filtrate (1) add 250 mL HAc-buff, adjust pH to 4.5 (2) 25℃ stir for 1 hours, (3) Filter Continued on next page … Continued from previous page … Filtrate Resin (to absorb protein) (1) add 40 g resin for adsorption (2) 25℃ stir for 1.5 h (3) wash with water Eluate (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 from previous page … Eluate Precipitation Add 3-time-volumn cold acetone Elastase powder (1) wash twice with acetone (2) vacuum drying End Product Obtained 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. 30 mg Congo red elastin(taken accurately) 1 2 20 mL standard elastic enzyme solution(must be sufficient) 100 mLvolumetric flask 3 Keep the flask at 37 ℃ in a water incubator 4 Shake the