Differential effects of intact subunits and nicked fragments of concanavalin A on immune functions in vitro and in vivo

Concanavalin A (Con A) is a widely used reagent in cell biology and immunology. Foremost among the many applications of this molecule are its use as a mitogen for T lymphocytes in vitro and to stimulate suppressor cells in vivo. Commercial preparations of Con A, including affinity-purified material, contain highly variable proportions of the intact protomeric subunit (m.w. 26.000) and the nicked fragments (m.w. 12.000 and 14.000) that result from proteolysis of the intact subunits within the Jack bean. The effect of this compositional heterogeneity on the biologic properties of Con A has not been investigated due to the lack of efficient procedures for separating nicked fragments from intact subunits. We have developed a method for efficient separation of intact and nicked subunits of Con A by using a combination of affinity chromatography and analytical high performance liquid chromatography (HPLC), which enables homogeneous preparations of intact and nicked subunits to be obtained on the scale needed for biologic assays. In this paper, we describe the effects of intact and nicked subunits of Con A on several in vitro and in vivo immune function assays in which this lectin is commonly employed. These studies indicate that differences in the proportion of 'contaminating' nicked Con A fragments present in commercial preparations of Con A are inconsequential for in vitro lymphocyte mitogenesis assays but represent a source of variation in experimental studies on antibody synthesis and suppressor cell induction in vivo.