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The reactions of human serum albumin (HSA) with optically active amino acid p-nitrophenyl esters (substrate, S) were examined kinetically at 25 degrees C. The rate data were analyzed in terms of a mechanism involving 1:1 complexing (S.HSA) between S and HSA. The dissociation constant (Ks in M) and the catalytic rate constant (k2 in s-1) of S.HSA were determined. Among ten substrates examined, the reactions with N-carbobenzoxy-D(L)-alanine p-nitrophenyl esters (N-CBZ-D(L)-AlaNP) were most accelerated by HSA. Results of the reaction in the presence of excess N-CBZ-D(L)-AlaNP over HSA indicated the existence of one strong reactive site on HSA. The effects of the reversible binding of the site-specific drug and the chemical modification by site-specific reagents on the HSA activity showed that the reactive site towards N-CBZ-D(L)-AlaNP is the R site located near tyrosine-411 residue of HSA.

The reaction of p-nitrophenyl 4-guanidinobenzoate (NPGB) with human serum albumin (HSA) was examined kinetically at various pH's and 25 degrees C. The Michaelis constant (Ks in M) and the catalytic rate constant (k2 in s-1) were determined. The ratio of k2 to k0 (hydrolysis rate constant of NPGB in s-1) at pH 7.4 was 75.6, indicating the esterase-like activity of HSA. The effects of the reversible binding of site-specific drugs and the chemical modification by site-specific reagents on the HSA activity indicated that HSA has multiple reactive sites towards NPGB. Results of the reaction in the presence of excess NPGB over HSA also suggested the existence of multiple active sites. The pH-profile for k2 showed inflection points at about pH 6.0 and pH 10.0, suggesting the involvement of groups with pKa's of 6.0 and 10.0 in HSA.