DANSYLALANYLLYSYLCHLOROMETHYL KETONE AS A FLUORESCENT-PROBE FOR LOCALIZATION OF ACROSIN ACTIVITY IN BOAR AND HUMAN-SPERMATOZOA

The localization of acrosin (EC 3.4.21.10) activity in mammalian spermatozoa was investigated by use of the fluorescent site-directed acrosin inhibitor, dansylalanyllysylchloromethyl ketone (DALCK). Fluorescence microscope preparations revealed, after the spermatozoa were subjected to a specific treatment, that acrosin activity is confined specifically to the inner acrosomal membrane (IAM). Spectrofluorometric and fluorescence polarization investigations verified that the fluorescent probe, once it is specifically bound to the treated spermatozoa, lies in a very hydrophobic environment and shows a remarkable reduction of rotational freedom. Under the experimental conditions used, active acrosin is tightly bound to the IAM and the specificity site of the acrosin-active center is probably of a highly hydrophobic nature.