Sulfhydryl Groups on the Spermatozoa Membrane. A Study with a New Fluorescent Probe for SH Group1

We report the properties of a new sulthydryl reagent, N˙ (4-carboxy-3-hydroxy-phenyl) maleimide (NCPM), which forms photostable, covalent fluorescent adducts with organic compounds, proteins and biological membranes containing sulfhydryl groups. NCPM appears not to permeate cell membranes and, since its reaction with SH compounds is stoichiometric, permits the quantitative assessment of the exposed functional groups on the surface of cells. Binding of NCPM to washed spermatozoa resulted in a considerable increase in fluorescence that was linearly related to the number of cells. Under similar experimental conditions rabbit sperm cells showed a significantly smaller binding of NCPM than human sperm cells (35 ± 5.2 against 55 ± 10 nmoles of SH groups/108 cells). This difference was enhanced in the presence of EDTA, which stimulated binding. Five times as high a concentration of SH groups was detected on the membrane of the sperm cells as on erythrocyte ghosts. In addition, while on the latter, most of the SH groups (85 percent) not readily reactive with NCPM, almost all of those present on the sperm cells are readily available.