ABSTRACT. We have previously reported the action of whey acidic protein (WAP) inhibiting the proliferation of mouse mammary epithelial cells in the experiments utilizing in vivo and in vitro systems. We report herein the bacteriostatic activity of WAP. Western blot analysis demonstrated successful isolation of WAP from whey fractions of rat milk by column chromatography. The WAP fraction inhibited the growth of Staphylococcus aureus JCM2413 in a dose-dependent manner, but did not inhibit the growth of Escherichia coli. The bacteriostatic activity of WAP was highest at pH 6.6 and was not affected by the presence of 150 mM NaCl. A scanning electron micrograph of bacteria treated with WAP exhibited the disruption of the bacterial cell walls. The whey acidic protein (WAP) with a four-disulfide core (4-DSC) structure (WAP motif) composed of eight cysteine residues has been identified in milk in a range of species [1,4,6,7,9,11,15]. Possible functions of WAP have been proposed based on its structural similarity to protease inhibitor [9]. However, only a few studies have attempted to determine the actual function of WAP [3,20]. We have previously reported that WAP inhibits the proliferation of mouse mammary epithelial cells in vivo and in vitro [12,[14][15][16][17] and has a function in reducing tumorigenesis and invasion of mammary cancer cells [18]. A number of antibacterial proteins with a WAP motif have been identified in a variety of tissues,-e.g., elafin [22], eppin [24], SLPI [25], SWAM1 and SWAM2 [10], although the molecular antibacterial mechanism of these proteins is not entirely clear. On the basis of homologous amino acid sequences in the WAP motif between WAP and known antibacterial proteins with WAP motifs, WAP may also have antibacterial potential. However, because the amino acid sequences of WAP motif proteins are widely divergent, with the exception of the conserved cysteine residues, it is quite difficult to predict their antibacterial activity merely by examining these sequences.In the present study, we investigated the antibacterial activity of WAP because there have been no studies investigating the effect of WAP on microbes.Milk was collected from lactating Wistar-Imamichi rats (Japan SLC, Hamamatsu, Japan) as described by Simoms et al. [21]. The viscous milk was diluted 1:3 with chilled distilled water, and fat was removed by centrifugation for 30 min at 3,100 g at 4C. The supernatant was adjusted to pH 4.6, and caseins were removed by centrifugation for 30 min at 3,100 g at 4C. The supernatant was adjusted to pH 7.0, and was then loaded on a Sephadex G-75 column (2.6 90 cm; Invitrogen, Carlsbad, CA, U.S.A.) equilibrated in 50 mM Tris-HCl (pH 7.0)/10 mM NaCl. Four milliliters of the eluate was collected at a flow rate of 0.2 ml/min at 4C and dialyzed overnight at 4C against distilled water. A part of each eluate was examined spectrophotometrically (SmartSpecTM Plus; Bio-Rad, Hercules, CA, U.S.A.) to determine the protein concentration using a protein assay kit (BioRad). The eluates wer...