Interaction of a Novel Class of Phospholipid-Binding Proteins of Bovine Seminal Fluid with Different Affinity Matrices

Abstract

We have recently shown that the major proteins from bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins) are novel phospholipid-binding proteins. These proteins show a Ca 2+-independent interaction with phosphorylcholine (PrC) containing lipids and this group acts as a specific binding site for BSP proteins on the sperm membrane. In this study, we investigated the biochemical basis of the binding of BSP proteins to the phosphorylcholine moiety using different affinity chromatography matrices containing active groups analogous to PrC. Proteins from bovine seminal fluid were applied to a column containing p-aminophenyl phosphorylcholine coupled to Agarose (PPC-agarose). Unadsorbed proteins were washed out with buffer (Tris-HCl) and elution of bound proteins was assessed with the same buffer containing different eluting agents. The adsorbed proteins were eluted with buffer containing specific (PrC or choline chloride), denaturing agent (urea), or chaotropic agent (sodium thiocyanate) and identified as BSP proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. At high ionic strength, these proteins could also be adsorbed on quaternary methylamine coupled to silica and diethylaminoethyl coupled to Sephadex and were eluted specifically with phosphorylcholine, choline chloride, or urea. These data suggest a structure-dependent hydrophobic interaction of the ligands with BSP proteins. Furthermore, binding experiments using equilibrium dialysis indicated that there are two choline binding sites per mole of protein. In addition, the combination of affinity chromatography columns and linear gradient of eluting agents allowed the complete fractionation of the different BSP proteins. The availability of an affinity chromatography method should aid in those studies aimed toward the understanding of the physiology of these phospholipid-binding proteins.