. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 130 N. PARRINELLO AND C. CANICATTI a b. 02 - - 16 2 4 ~ ~ E o ai aj I i 120 E lution volu me (ml) 140 160 200 FIGURE 2. Affinity chromatography of Phallusia mamil/ata hemagglutinins on acid-treated Se- pharose CL-6B. PBS-dialyzed serum (20 ml) was applied to a column ( X 15 cm) at 25°C. The column was washed with 1 M NaCl and, at the point indicated, elution was started with 1 M NaCl plus M D-galactose. The flow rate was 20 ml/h. The eluted fractions were dialyzed against NaCl (1 M) and PBS to remove the sugar. All th
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 130 N. PARRINELLO AND C. CANICATTI a b. 02 - - 16 2 4 ~ ~ E o ai aj I i 120 E lution volu me (ml) 140 160 200 FIGURE 2. Affinity chromatography of Phallusia mamil/ata hemagglutinins on acid-treated Se- pharose CL-6B. PBS-dialyzed serum (20 ml) was applied to a column ( X 15 cm) at 25°C. The column was washed with 1 M NaCl and, at the point indicated, elution was started with 1 M NaCl plus M D-galactose. The flow rate was 20 ml/h. The eluted fractions were dialyzed against NaCl (1 M) and PBS to remove the sugar. All the fractions were assayed for hemagglutinating activity with RE and HE, hemagglutinating tilers were expressed as reciprocal of the serum dilution. Inset: developed portions of the polyacrylamide gels showing electrophoretic patterns; a) whole serum, b) pooled D-galactose eluted fraction showing maximum hemagglutinating titer. Arrow indicates the origin. the gel bed. The first peak observed in the elution profile contains all the inactive proteins. The column was then washed with 1 M NaCl to elute the non-agglutinating fractions from Sepharose. Finally, when the eluate had reached a constant low absorbance at 280 nm, anti-RE and anti-HE agglutinins were displaced from the Sepharose bed by a solution of D-galactose ( M) in 1 M NaCl. These experiments clearly demonstrate that M D-glucose in 1 M NaCl so- lution, a non-inhibitor of the agglutinin-polysaccharide interaction, did not elute agglutinins specifically bound to Sepharose, whereas a-lactose, like D-galactose, ( M solution containing 1 M NaCl) readily displaced them from the gel bed. NaCl solution (1 M) or sugar in PBS ( M solution) were unable to elute the agglutinins. Electrophoretic and immunological characterization of the isolated agglutinins The whole serum and the D-galactose-eluted protein were investigated by poly- acrylamide gel electrophoresis. In Figure 2 the patterns of the P. mamillata serum (Fig.
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
