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

Interaction of camel lens zeta -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-zeta -crystallin binding. Quantitative analysis indicated that 1 mole zeta -crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace zeta -crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in zeta -crystallin, possibly at the NADPH binding site, alpha -Crystallin as well as NADPH protected zeta -crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of zeta -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 muM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K 'I) of 12 muM for the zeta -crystallin-NADPH-ANS complex. The Ki being smaller than K 'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-zeta -crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time. (C) 2001 Elsevier Science B.V. All rights reserved.