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  • 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 1Purposes2Part I: Preparation of Elastic Protease3Part II: Analysis of Enzyme Activity 3. Purposes1Learn the principle of preparation of elastic protease from pancreas.2Master the analysis method of enzyme activity. 4. Part IPreparation 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 Continued on Filtrate Filtrate next page next page 6. Procedures II Continued from Continued from Filtrate Filtrate previous page previous page (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 Continued on Continued on Eluate Eluate next page next page 7. Procedures III Continued from Continued from Eluate Eluate previous page previous page Add 3-time-volumn cold acetone Precipitation Precipitation (1) wash twice with acetone (2) wash once with ether (3) vacuum drying Elastase powder Elastase powderEnd Product Obtained 8. Part IIAnalysis 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 I1 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 II5 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 scale3 Keep the flask at 37 in a water incubator 12. Sample Preparation12 5 mL pH 8.8 boric acid buff (add a small amount at first)5 mg enzyme sample (taken accurately)mortar3 grind until everything dessolves1 mLboric acid buff4 Dilute enzyme solution with boric acid buff to 2 ~ 3 unit/mL 13. Standard Curve1 2Take 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 No012345Reference substance (mL)02.04.06.08.010.010.08.06.04.02.00Mixture 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)0121.233544Await 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. Caculation1Take the average absorption and check the corresponding enzyme activity according to the standard curve.2Calculate the relative enzyme activity (enzyme activity/enzyme amount) by taking into account the dilution times.3Calculate the total enzyme recovery yield. 16. The EndIts time for your practice. EVERYBODY GO GO GO! PPT 2011.9