Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes BCMB 301 A LAB 6B:

1. Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes BCMB 301 ALAB 6B:

2. Trypsin and α-Chymotrypsin • Proteolytic Enzymes • Serine Proteases • Synthetic Substrates • Inhibitors

3. Trypsin and α-Chymotrypsin • Proteolytic Enzymes • Serine Proteases • Synthetic Substrates • Inhibitors

4. Trypsin and α-Chymotrypsin • Proteolytic Enzymes • Serine Proteases • Synthetic Substrates • Inhibitors • BAPNA & GPPNA • Structurally resemble tripeptides • Enzymatic cleavage produces p-nitro anilide. Detectable at A405nm

5. Trypsin and α-Chymotrypsin • Proteolytic Enzymes • Serine Proteases • Synthetic Substrates • Inhibitors E + I E….I E-I’

6. REAGENTS ARE POISONOUS. WEAR GLOVES.

7. A. Substrate Specificity Prepare 3 cuvettes • Buffer: assay buffer • Enzyme: trypsin or chymotrypsin • Substrate • BAPNA • GPPNA • 100% DMF SET REF Measure A405nm every 15 seconds for 3 min

8. B. Inhibitor Specificity Prepare 5 microfuge tubes: Incubate Into a new microfuge tube: **from previous microfuge tubes SET REF Measure A405nm every 15 sec for 3 min WASH YOUR HANDS. THERE IS POISON ON THEM.

9. Results Exchange data with your partner

10. Results Part A • 2 graphs • One for trypsin, one for chymotrypsin Part B • 2 graphs • Table: % inhibition for each inhibitor • % inhibition = |Vuninh – Vinh| * 100 Vuninh

11. Discussion • Number the questions • Be concise • Answer the questions

12. Colored Eppendorf Tubes BAPNA – blue GPPNA – pink Trypsin – purple Chymotrypsin - yellow