Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes BCMB 301 A LAB 6B:
12
Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes BCMB 301 A LAB 6B:
description
Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes BCMB 301 A LAB 6B:. Trypsin and α -Chymotrypsin. Proteolytic Enzymes Serine Proteases Synthetic Substrates Inhibitors. Trypsin and α -Chymotrypsin. Proteolytic Enzymes Serine Proteases Synthetic Substrates Inhibitors. - PowerPoint PPT Presentation
Transcript of Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes BCMB 301 A LAB 6B:
Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes
BCMB 301 ALAB 6B:
Trypsin and α-ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitors
Trypsin and α-ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitors
Trypsin and α-ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitors
BAPNA & GPPNA
Structurally resemble tripeptides
Enzymatic cleavage produces p-nitro anilide. Detectable at
A405nm
Trypsin and α-ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitors
E + I E….I E-I’
REAGENTS ARE
POISONOUS.
WEAR GLOVES.
A. Substrate Specificity
Prepare 3 cuvettes
Buffer: assay bufferEnzyme: trypsin or chymotrypsinSubstrate
BAPNAGPPNA100% DMF
SET REF
Measure A405nm every 15 seconds for 3 min
B. Inhibitor Specificity
Prepare 5 microfuge tubes:Incubate
Into a new microfuge tube:**from previous microfuge tubes
SET REFMeasure A405nm every 15 sec for 3 min
WASH YOUR HANDS. THERE IS POISON ON THEM.
Results
Exchange data with your
partner
Results
Part A2 graphs
One for trypsin, one for chymotrypsin
Part B2 graphsTable: % inhibition for each inhibitor
% inhibition = |Vuninh – Vinh| * 100 Vuninh
Discussion
Number the questions
Be conciseAnswer the
questions
Colored Eppendorf Tubes
BAPNA – blueGPPNA – pinkTrypsin – purpleChymotrypsin - yellow
