Purification of P26h: a hamster sperm protein

P26h is a 26 kDa glycoprotein, located on the acrosome cap of hamster spermatozoa, involved in the species specificity of gamete interaction. We have purified this protein from hamster spermatozoa collected from the distal cauda region of the epididymis. Its purification was realized following a three-step procedure: detergent extraction, ion-exchange chromatography, and chromatofocusing. Protein partitioning using Triton X-114 (the first step) showed a ratio of 5:1 between the resulting aqueous and detergent phase. P26h was found almost exclusively in the aqueous phase where it represented about 10-12% of the total protein content. When the desalted aqueous phase was loaded on a carboxymethyl column for cation-exchange chromatography, about 80% of the proteins did not bind to the matrix and were eliminated. P26h was eluted from the column with a linear gradient of salt. At this point, P26h had a rate of purification estimated at 45-55%; three other major proteins of <21, 45, and 63 kDa remained in the sample. These undesired proteins were eliminated by submitting these samples to chromatofocusing using a PBE 94 polybuffer exchanger column. Indeed, P26h was collected almost in the dead volume of the column while the other proteins were eluted much later. Two-dimensional gel electrophoresis and Western blotting were performed to determine the purity of P26h. Only one major spot was detected, confirming the purity of P26h. Usefulness of this purified sperm antigen in the understanding of the physiology of mammalian fertilization is discussed.