. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 46 B. M. CHAIN AND R. S. ANDERSON st CF .. FIGURE 4. Antibacterial activity in coelomic fluid (CF) visualized after electrophoretic fractionation on a polyacrylamide gradient gel. a) Area of a gel overlayed with bacteria (6 x 105 S. marcescens/m\ in TS agar). b) Portion of the same gel stained for protein with Coomassie blue. Protein standards (st) are the same as used in Figure 3; A = Major protein band of CF. diately above it, and the other to a very high molecular weight component. It seemed likely that this second band o
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 46 B. M. CHAIN AND R. S. ANDERSON st CF .. FIGURE 4. Antibacterial activity in coelomic fluid (CF) visualized after electrophoretic fractionation on a polyacrylamide gradient gel. a) Area of a gel overlayed with bacteria (6 x 105 S. marcescens/m\ in TS agar). b) Portion of the same gel stained for protein with Coomassie blue. Protein standards (st) are the same as used in Figure 3; A = Major protein band of CF. diately above it, and the other to a very high molecular weight component. It seemed likely that this second band of inhibition resulted from aggregation of GAP which occurs under experimental conditions and probably corresponds to S-300 protein peak 1. Other purification methods Ion exchange chromatography on DEAE-Sepharose anionic columns (at pH ) was carried out using linear ionic strength elution gradients. Although excellent separation of several components was obtained, no activity could be recovered. Once again this could not be attributed to separation of two or more components required for activity since elution of all components simultaneously (using a one-step elution change from low ionic strength to ASW) still resulted in total loss of activity. Simple passage through the column at ionic strengths too high to allow binding had no effect on activity. It would appear that the interactions occurring during binding of coelomic fluid components to the column or during dilution, were sufficient to permanently destroy the bactericidal activity. A more successful approach was affinity chromatography on a Con A Sepharose 4B column (Fig. 5). The majority of the coelomic fluid components failed to bind to the column and could be eluted with ASW. However, all antibacterial activity was adsorbed onto the column. A fraction eluted from the column with a high concentration of a-methyl D-glucopyrannoside (or mannoside) showed a bacteri- cidal effect. However, this activity represented only
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