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

National Animal Disease Center, Agricultural Research Service, US Department of Agriculture, P.O. Box 70, Ames, IA 50010

ABSTRACT

Previously three factors (F–1, F–2, F–3) that stimulated in vitro growth of Streptococcus agalactiae were separated from wheys of milk and colostrum by chromatographic procedures. Now F–1 was separated further into two 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 two protein peaks whereas a third protein peak that was eluted in those fractions containing lactose was not stimulatory. Most of the stimulatory activity (76 to 80%) and protein (90 to 93%) were retained by a filter with retention of > 1,000 MW.

Two stimulatory factors were confirmed by separation of F–1 fractions by step-gradient elution on a column of octadecylsilica; one protein peak was found for each of the three solvents. Samples with protein eluted with .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 (µBondapack 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 two 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 one or more of the proteose-peptones in whey.