ELECTROPHORETIC ANALYSIS OF PROTEIN SELECTIVELY EXTRACTED WITH CHAOTROPIC AGENTS FROM MEMBRANES OF RAT SPERM

Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. Th e 100.000 x g fraction contained only small membranous vesicles and le ss than 0.5% of the total enzymatic activity. This fraction was consid ered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetat e buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-10 0, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2% SDS. The extracts and the solubil ized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtai ned with the first three chemicals contained 11, 9, and 25% of total p roteins respectively. The bicarbonate buffer solubilized 45%, and the detergents 55% and 65% respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbon ate buffer, extracted a great number of bands over a wide range of mol ecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had sim ilar effects: both solubilized four new bands. In residual sediments t here were no new proteins and the bands corresponded to those extracte d with the detergents, but they varied in staining intensity. Accordin g to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50% of the mass of membraneous proteins may b e peripheric. Proteins partially extracted with the detergents were al so found in the residual sediment, and they may constitute the skeleto n of sperm membrane.