Quenching of intrinsic fluorescence of sperm specific LDH by optical isomers of gossypol.

Intrinsic fluorescence of LDH-C4 has been studied in the presence of optical isomers of gossypol. The study showed that fluorescence due to tryptophan residues after excitation of LDH at 282 nm is quenched by each gossypol enantiomere in a concentration dependent manner. Half of the maximum quench (Q50%) of enzyme occurred with gossypol (-) at 0.9 x 10(-4) mol/l and with gossypol (+) at 1.4 x 10(-4) mol/l showing a maximum quench (Qmax) of 45% and 65% respectively, with a corresponding association constant (Ka) of 1.0 x 10(4) l/mol and 0.4 x 10(4) l/mol. Stern-Volmer constant (Ksv) inferred that quenching of LDH comprises at least two components with two different Ksv values. Ksv(I) and Ksv(II) between LDH-C4 and gossypol (-) were 1.97 x 10(3) l/mol and 1.22 x 10(3) l/mol, and those between LDH-C4 and gossypol(+) were 2.3 x 10(3) l/mol and 1.56 x 10(3) l/mol. Smaller Ksv at higher concentrations of gossypol indicated that some of the tryptophan residues in LDH-C4 are deeply buried within a hydrophobic environment. There was no blue or red shift of LDH-C4 when interacting with either of the gossypol enantiomeres.