A new type of Phaseolus vulgaris (cv. Pinto III) seed lectin: Isolation and characterization

Abstract

From the seeds of Phaseolus vulgaris cv. ‘Pinto III’, previously regarded as a hemagglutinin-free bean, several glycoprotein lectins were purified by conventional methods, including solubility- and salt-fractionation, continuous high-voltage free-flow electrophoresis and molecular sieve chromatography. A preparation of similar lectins was also obtained by immunoaffinity chromatography on a Sepharose-4B column to which rabbit anti-Pinto seed lectin (conventionally purified) immunoglobulins had been attached. Both preparations gave one band of 28 000–29 000 subunit weight on SDS-polyacrylamide gel electrophoresis. They were however shown to contain components, with isoelectric points in the range of pH 4.7–5.0, by isoelectric focusing on polyacrylamide gels. The relative proportion of the individual lectin components was slightly different in the two preparations. Their sedimentation coefficient, 4.34 S, was the same; however, they had a slight difference in partial specific volume values; 0.691 ml/g for the conventional and 0.700 ml/g for the affinity preparation. The average molecular weight value ($\text{M}{}_{\mathrm{<mtext>r,av</mtext>}}{}^0$) of 52 200 for the conventional preparation was significantly lower ($\text{P > 0.1}$) than the value of 55 000 for the affinity-purified lectin. The ‘Pinto III’ seed lectin molecules contained two subunits only in place of the usual four subunits of the common P. vulgaris lectins. There was a slight immunochemical cross-reaction between the common P. vulgaris and the ‘Pinto III’ seed lectins. The ‘Pinto III’ seed lectins had low haemagglutinating activity when tested with rabbit erythrocytes, while their activity was high against pronase-treated rat cells. It is suggested that the low activity against rabbit cells might be the result of the lower affinity of the dimeric lectin for the exposed sugar structures on the red cell membrane.