Effect of lysine modification on the conformation and indomethacin bindingproperties of human serum albumin

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which r esulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtr ation on a Seralose-6B column. Hydrodynamic and circular dichroic results s howed that pronounced conformational changes (both tertiary and secondary s tructures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in alpha-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 8 6% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, res pectively) was also noticed in these highly modified preparations. Fluoresc ence quench titration results obtained at pH 7.4 and ionic strength 0.15 sh owed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respe ctively, as compared to 91% retention in binding in 86% guanidinated prepar ation. No reversal in the binding of drug to 87% acetylated and 78% succiny lated HSA preparations was observed on increasing the ionic strength to 1.0 . Therefore, it seems that one or two critical lysine residue(s) that can f orm salt linkage with the carbo?;yl group of indomethacin, was (were) proba bly modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indome thacin to HSA. (C) 1999 Published by Elsevier Science B.V. All rights reser ved.