Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library.
In this paper we present an HPLC method developed for quick activity and specificity analysis of serine proteinases. The method applies a carefully designed peptide library in which the individual components differ only at the potential cleavage site for enzymes. The library has seven members representing seven different cleavage sites and it offers substrates for both trypsin and chymotrypsin-like enzymes. The individual peptide substrates compete for the proteinase during the enzymatic reaction. The reaction is monitored by RP-HPLC separation of the components. We describe the systematic design of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the investigated enzymes as determined by the new method were essentially identical to the ones reported in the literature, verifying the ability of the system to characterize substrate specificity. The tests also demonstrated that the system could detect even subtle specificity differences of two isoforms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding specificity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering.
Antal
J
J
Analysis-Synthesis Center, Agricultural Biotechnology Center, Szent-Györgyi A. út 4, Gödöllo, H-2100, Hungary. antal@abc.hu
Pál
G
G
Asbóth
B
B
Buzás
Z
Z
Patthy
A
A
Gráf
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009842:Oligopeptides / Q000378:metabolism*; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D009842:Oligopeptides; D019151:Peptide Library; D012697:Serine Endopeptidases