[Isolation of low-molecular-mass antibacterial mixtures from human seminal plasma]

To isolate low-molecular-mass antibacterial mixtures from healthy human seminal plasma.

Semen was obtained by masturbation after at least three days of abstinence from healthy donors. Semen samples were allowed to liquefy at room temperature and then centrifuged at 10,000 r/min for 10 min to separate spermatozoa from seminal plasma. High sensitive antimicrobial activity was measured with radial diffusion assay. Antibacterial activity toward E. coli (ATCC25922) was monitored for each purification steps. The mixture of seminal plasma samples was applied to a SP-Sepharose column. Fractions which showed strong bactericidal activities, were combined and lyophilized. The lyophilized components were dissolved with Milli-Q water and applied to AKTA Superdex 75 column. Peak II of the Superdex 75 column, which showed antibacterial activity and represented the low-molecular-weight cationic fractions of the seminal plasma, was collected and lyophilized. Finally, peak II of the Superdex 75 column was applied to reverse phase HPLC C18 column. Fractions which showed strong antibacterial activity, were lyophilized and store at -20 degrees C. The molecular weight of the low-molecule-mass antibacterial mixtures was determined by Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.

The low-molecular-mass mixtures with obviously higher antibacterial activity, which were termed HSLAMs(Human semen low-molecular-mass antibacterial mixtures), were isolated from the healthy human seminal plasma. Based on the mass spectrometry results, some molecules of RP-HPLC peaks were confirmed to be the semenogelin I derived peptides.

The low-molecule-mass antibacterial mixtures may play an important role in males innate immunity. Semenogelin I derived peptides may be one of the sources of the low-molecule-mass antimicrobial mixtures in human seminal plasma.