Sequential inactivation of zeta-crystallin by o-phthalaldehyde.
o-Phthalaldehyde, a bifunctional cross-linking reagent, is commonly used as a probe for the active site of enzymes. In this study, the interaction of o-phthalaldehyde with camel lens zeta-crystallin was examined by activity and fluorescence measurements. Predictably, the oxidoreductase activity of zeta-crystallin was inhibited irreversibly by o-phthalaldehyde in a time- and concentration-dependent manner, and the presence of NADPH with the enzyme appeared to provide a high degree of protection against o-phthalaldehyde inactivation. Interaction of o-phthalaldehyde with zeta-crystallin resulted in formation of isoindole adduct, which exhibited characteristic fluorescence at 415 nm. However, neither inactivation nor modification of the enzyme showed the expected pseudo-first-order kinetics; both events were highly sequential reaching different levels of saturation at different concentrations of o-phthalaldehyde. The modified enzyme had a maximum stoichiometry of 1 mol isoindole/subunit, and bound NADPH to nearly the same extent as unmodified enzyme. Gel filtration experiments suggested that o-phthalaldehyde-modified zeta-crystallin had higher apparent molecular weight than unmodified enzyme, even though the enzyme remained largely monomeric as revealed by electrophoresis on denaturing gel. These results suggested that modification by o-phthalaldehyde might have been so intrusive as to sequentially modify the tetrameric structure of zeta-crystallin.
Bazzi
Mohammad D
MD
Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. mbazzi@ksu.edu.sa
Rabbani
Nayyar
N
Duhaiman
Ali S
AS
D000818:Animals; D001665:Binding Sites; D002162:Camelus; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003459:Crystallins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D009764:o-Phthalaldehyde / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D003459:Crystallins; D009249:NADP; D009764:o-Phthalaldehyde; D010088:Oxidoreductases