Fractionation of a Whey Growth Factor for Steptococcus agalactiae into Two Active Components Containing Proteins

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Abstract
Previously, 3 factors (F-1, F-2, F-3) that stimulated in vitro growth of S. agalactiae were separated from wheys of [bovine] milk and colostrum by chromatographic procedures. Now F-1 was separated further into 2 active components, F-1a and F-1b, when rechromatographed on an anion exchange resin with distilled water as eluent. The stimulatory activity was associated with 2 protein peaks, whereas a third protein peak that was eluted in those fractions containing lactose was not stimulatory. Most of the stimulatory activity (76-80%) and protein (90-93%) were retained by a filter with retention of > 1000 MW. Two stimulatory factors were confirmed by separation of F-1 fractions by step-gradient elution on a column of octadecylsilica; 1 protein peak was found for each of the 3 solvents. Samples with protein eluted with 0.1 N formic acid and formic acid:methanol (1:1) had stimulatory activity, whereas the sample with protein eluted with 100% methanol was inactive. Results were similar when F-1 fractions were applied to Sep-Pak (.mu.Bondapack C18) cartridges and eluted with water, 50% methanol and 100% methanol. Proteins in the Sep-Pak eluates with stimulatory activity (water and 50% methanol) were heterogeneous and different when separated by high-voltage paper electrophoresis. Proteose-peptone preparations with added lactose were chromatographed on columns of anion exchange resin, with distilled water as eluent, and produced 2 peaks of protein with elution volumes comparable to peaks 1 and 3. Lactose appeared to be a major factor in eluting the second protein peak. The F-1a and F-1b might be peptide fragments of 1 or more of the proteose-peptones in whey.