A microtiter plate assay for the characterization of serine proteases by their esterase activity.
The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which X is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96-well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, alpha-lytic protease, subtilisin, and proteinase K were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means.
Whittaker
R G
RG
CSIRO Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, New South Wales, Australia.
Manthey
M K
MK
Le Brocque
D S
DS
Hayes
P J
PJ
D002918:Chymotrypsin / Q000378:metabolism; D019286:Endopeptidase K; D004950:Esterases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D010196:Pancreatic Elastase / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D007202:Indicators and Reagents; D004950:Esterases; D012697:Serine Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; C021826:myxobacter alpha-lytic proteinase; D010196:Pancreatic Elastase; D014357:Trypsin; D013917:Thrombin; D019286:Endopeptidase K