High-affinity binding of NADPH to camel lens zeta-crystallin.
Fluorescence spectrum of camel lens zeta-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including alpha-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with zeta-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and zeta-crystallin with an apparent Km<0.45 microM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of zeta-crystallin, also was able to quench the fluorescence of zeta-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that zeta-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in zeta-crystallin.
Bazzi
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MD
Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, 11451, Riyadh, Saudi Arabia.
Rabbani
N
N
Duhaiman
A S
AS
D000818:Animals; D002162:Camelus; D003459:Crystallins / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D009249:NADP