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