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 fro m the distal cauda region of the epididymis. Its purification was real ized following a three-step procedure: detergent extraction, ion-excha nge chromatography, and chromatofocusing. Protein partitioning using T riton X-114 (the first step) showed a ratio of 5:1 between the resulti ng aqueous and detergent phase. P26h was found almost exclusively in t he aqueous phase where it represented about 10-12% of the total protei n content. When the desalted aqueous phase was loaded on a carboxymeth yl column for cation-exchange chromatography, about 80% of the protein s 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 o f <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 almo st in the dead volume of the column while the other proteins were elut ed much later. Two-dimensional gel electrophoresis and Western blottin g were performed to determine the purity of P26h. Only one major spot was detected, confirming the purity of P26h. Usefulness of this purifi ed sperm antigen in the understanding of the physiology of mammalian f ertilization is discussed.