. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. POLYCHAETE ANTIBACTERIAL FACTOR, II. 47 20 •J 40 B 60 o DQ 80 100. h-ASW elution Sugar elution FIGURE 5. Affinity chromatography of CF on Con A-Sepharose 4B. Ten ml of coelomic fluid were run through a short (8 X cm) column of Con A-Sepharose using artificial sea-water as the eluent. At arrow eluent was changed to a M solution of 1-0-methyl-a-D-glucopyranoside in ASW. Three ml were run onto the column and flow was stopped for 30 min to allow time for the competing ligand to displace substances bound by the Con A. Gly
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. POLYCHAETE ANTIBACTERIAL FACTOR, II. 47 20 •J 40 B 60 o DQ 80 100. h-ASW elution Sugar elution FIGURE 5. Affinity chromatography of CF on Con A-Sepharose 4B. Ten ml of coelomic fluid were run through a short (8 X cm) column of Con A-Sepharose using artificial sea-water as the eluent. At arrow eluent was changed to a M solution of 1-0-methyl-a-D-glucopyranoside in ASW. Three ml were run onto the column and flow was stopped for 30 min to allow time for the competing ligand to displace substances bound by the Con A. Glycoproteins were then eluted with more sugar solution. At second arrow eluent was changed back to ASW. Three ml fractions were collected throughout the ex- periment and ml of each fraction tested for bactericidal activity using the turbidometric assay. Note that the high sugar concentration alone inhibited bacterial growth to a certain extent. - UV absorbance (280 nm) - Bactericidal activity activity put onto the column, either because the active factor remained bound to the column, or because it had been irreversibly denatured by binding to the column. Figure 6a shows an analysis of the Con A fractionation using polyacrylamide gradient gel electrophoresis. Although Con A selectively removes certain high mo- lecular weight components of the coelomic fluid, most of the major proteins in the region of band A are not retained, suggesting that GAP is quantitatively a very minor constituent of CF proteins of this molecular weight. However, electrophoresis of a concentrate of the material initially bound to Con A, and subsequently eluted with methyl glucose, showed that this fraction indeed included a small component of the major band (A). Since Con A selectively binds glycoproteins, samples of whole CF and the Con A binding fraction were also stained with the PAS glycoprotein stain (Fig. 6b). The PAS-positive bands in whole CF correspond rather closely to those bound to Con A.
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