Hydrophobicity of the NADPH binding domain of camel lens ζ-crystallin

摘要

Interaction of camel lens ζ-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-ζ-crystallin binding. Quantitative analysis indicated that 1 mole ζ-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace ζ-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in ζ-crystallin, possibly at the NADPH binding site. α-Crystalline as well as NADPH protected ζ-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH: quinone oxidoreductase activity of ζ-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K_i of 2.3 μM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K'I) of 12 μM for the ζ-crystallin-NADPH-ANS complex. The K_i being smaller than K'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-ζ-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.