Effect of lysine modification on the conformation and indomethacin binding properties 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 resulted 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 filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) 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 86% 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, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed 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, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated 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 form salt linkage with the carboxyl group of indomethacin, was (were) probably 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 indomethacin to HSA.
Tayyab
S
S
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, U.P. India. btisamu@x400.nicgw.nic.in
Haq
S K
SK
Sabeeha
Aziz
M A
MA
Khan
M M
MM
Muzammil
S
S
D000089:Acetic Anhydrides / Q000737:chemistry; D001665:Binding Sites; D002317:Cardiovascular Agents / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007213:Indomethacin / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008782:Methylurea Compounds / Q000737:chemistry; D009994:Osmolar Concentration; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D013387:Succinic Anhydrides / Q000737:chemistry
D000089:Acetic Anhydrides; D002317:Cardiovascular Agents; D008782:Methylurea Compounds; D012709:Serum Albumin; D013387:Succinic Anhydrides; C002249:O-methylisourea; C031800:acetic anhydride; C031801:succinic anhydride; D008239:Lysine; D007213:Indomethacin