Characterization of N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide for the detection of zinc in living sperm cells

Abstract

Zinc stabilizes membranes and DNA and inhibits respiration in somatic cells. It is present in high concentrations in the male reproductive tract and may stabilize spermatozoa prior to fertilization. Herein, we evaluate N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) for analysis of Zn2+ in phosphatidylcholine (PC) vesicles and living spermatozoa using spectrofluorometry and flow cytometry. TSQ:Zn fluorescence in decanol or PC vesicles was compared to that in aqueous buffer. Evaluation of cation specificity, kinetics of TSQ:Zn binding, quenching of TSQ by dithionite and Zn2+ chelation by D-penicillamine established that TSQ is more fluorescent in decanol or PC vesicles than in aqueous buffer, has a high affinity for lipid bilayers and is specific for Zn2+ compared to Mg2+ and Ca2+. Fluorescence measurement of vesicles with and without pretreatment with Zn2+ indicated that, in the absence of Zn2+, 90% of the residual TSQ fluorescence was destroyed by dithionite but > 50% was protected by the presence of Zn2+. When D-penicillamine was added the remaining fluorescence was quenched (T1/2 = 10 s) indicating that TSQ remains in/on the membrane. These results established that TSQ can be used to effectively evaluate Zn2+ in artificial membranes and sperm cells. Additional experiments will be necessary to explain the dynamics of TSQ:Zn:membrane interactions.