Ferritin labeling of concanavalin A by glutaraldehyde. Comparison of different molar con A/ferritin/ glutaraldehyde ratios and characterisation of the products

Abstract

Optimal conditions were investigated for preparation and isolation of monomeric Con A ferritin, with one molecule ferritin bound to one tetravalent molecule of Con A. Con A and ferritin were covalently coupled with highly purified glutaraldehyde as a bifunctional reagent at different molar ratios. The effect of four different concentrations of glutaraldehyde and of two different ratios of 125I-Con A to ferritin on the yield of Con A-ferritin monomers were tested in one-step reactions. The different products were subjected to linear sucrose density gradient centrifugation and the amount of 125I-Con A or ferritin or conjugates in the fractions obtained were measured by optical density at 280 nm (protein) and 310 nm (ferritin), gamma-counting ( 125I-Con A) by quantitative immunoelectrophoretic techniques (free, ferritin, ferritin bound to Con A), and by counting relative numbers of ferritin monomers, dimers, trimers and larger oligomers under the electron microscope. In the 30–56% w/w sucrose gradient unlabeled Con A was optimally separated from ferritin or ferritin-labeled Con A monomers. Ferritin monomers and Con A ferritin monomers could be recovered at a sucrose concentration of 42 to 49% w/w. A high molar ratio of Con A to ferritin during the coupling reaction caused formation of high molecular weight Con A-polymers. Optimal yield of Con A-ferritin monomers was obtained with an equimolar ratio of Con A to ferritin, a glutaraldehyde to ferritin ratio of 26 : 1 and a final ferritin concentration of 310 nmoles/ml.