Harris-Smith & Stanley, 1951) has shown that the virulence-enhancing action of hog gastric mucin is due to a synergic action between two non-specific factors (a viscous medium and a particulate residue) and a more specific soluble 'third factor'. Partial purification of the latter (Smith, 1951) yielded a heterogeneous product which was predominantly peptide in nature, but which contained 5-10 0 % of carbohydrate residues. This paper describes further purification of the third factor using a revised assay (Smith et al. 1951) in which the two non-specific factors are maintained constant. It has been shown that the carbohydrate moiety is responsible for the activity. Furthermore, this moiety has been separated into at least two active fractions, which have different chemical and biological properties, and which, when combined together, show a synergic effect in the system of the revised assay. This is an important result affecting investigations on the mode of action of mucin. In the light of this work, various bacterial polysaccharides have been examined for virulenceenhancing activity in the assay for the third factor: they are active and the results are included in this paper, together with a report on their other relevant biological properties. EXPERIMENTAL AND RESULTS Methods Activities are given in 'Virulence-enhancing units' (v.E.u.) (Smith et al. 1951) by direct comparison with a standard mucin, with fiducial limits shown in brackets for P = 0.95. The yields and activities of samples obtained from a particular fractionation are quoted for a single experiment. The activities of the various fractions may not be significantly different when taken alone, but become so when coupled with similar results in a number of such fractionations on different batches of the third factor. The inclusion of all the latter results would be superfluous. Hexosamine, which had been liberated from the various polysaccharides by hydrolysis with HC1 at 1000 in sealed tubes, was estimated by the method of Elson & Morgan (1933) using the NaHC03-Na2008 buffer of Immers & Vasseur (1950) for the acetonylacetone condensation. Replicate assays on different hydrolysates showed the method to have an error of ± 5 % for most ofthe compounds used (see also Wolfrom, Weisblat, Karabinos, McNeely & McLean, 1943; Meyer, 1945). Uronic acid was determined by the carbazole method of Dische (1947), and by the modification due to Tracey (1948) of the classical Lefevre & Toilens (1907) method. Discrepancies between the two methods for heparin and chondroitin sulphuric acids have been noted by Dische (1947). Replicate assays showed that the error of the Dische method was ±5%, and of the classical method
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