Preparation and Properties of Metal Ion Derivatives of the Lentil and Pea Lectins

Lentil lectin (LcH) and pea lectin (PSA) belong to the class of d-glucose/d-mannose binding lectins and resemble concanavalin A (Con A) closely in physicochemical, structural, and biological properties. LcH and PSA, like Con A, are Ca2+-Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. Studies of the relationship between the metal ion binding and saccharide binding activity in LcH and PSA have been difficult due to the problem of metal ion replacement in these proteins. We now report a method of metal ion replacement in both lectins that allows substitution of the Mn2+ in the native proteins with a variety of transition metal ions, as well as substitution of the Ca2+ with Cd2+ in a particular complex. The following metal ion derivatives of both LcH and PSA have been prepared: Ca2+-Zn2+, Ca2+-Co2+, Ca2+-Ni2+, and Cd2+-Cd2+. All of these derivatives are as active as the native lectins, as demonstrated by precipitation with specific polysaccharides, saccharide inhibition of precipitation, and hemagglutination assays. The yields of these derivatives are good (generally >70%), and the degree of metal ion incorporation is high (generally >90%). The method of preparation is quite different from that for metal ion substitution in Con A, which proceeds via the apoprotein. In contrast, the apoproteins of LcH and PSA are unstable, aggregate above pH 4.0, and cannot be remetallized once formed. In general, we have found that equilibration of either lectin in acetate buffer at pH 4.0, 37 °C, with 0.1 M of the appropriate metal salt plus 0.1 M CaCl2, leads to the corresponding Ca2+-M2+-substituted derivative of both proteins, after approximately 2 weeks for LcH and 4 weeks for PSA. Only Cd2+ leads to replacement at both Mn2+ and Ca2+ sites. Evidence indicates that metal ion release and capture by LcH and PSA are very different from those of Con A, which are sequential. By a number of criteria, the intrinsic molecular properties of these metal ion derivatives of both lectins are the same as those of the native proteins.