Electrophoretic map of acidic and neutral human spermatozoal proteins

A high resolution two dimensional electrophoretic technique (2-D), isoelectric focusing followed by polyacrylamide gel electrophoresis, for separation of neutral and acidic human spermatozoal proteins was established. Silver stained 2-D gels revealed over 260 proteins with Mr 20-200 kDa and pI between 4.5 and 7.8. Membrane proteins were radio-iodinated in their hydrophobic cores with the hydrophobic photoactivatable reagent 3 (trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine (TID). Externally exposed carbohydrate side chains were radiolabelled by NaB(3H)4 reduction. 2-D lectin blotting was performed with horseradish peroxidase conjugated Concanavalin A (Con A), biotinylated Vicia faba agglutinin (VFA) and Solanum tuberosum agglutinin (STA). A map of 258 human spermatozoal proteins is presented and 48 TID-labelled proteins have been biochemically characterized.