POLYCLONAL IMMUNOGLOBULIN-M - LOCATION OF GLYCATION SITES

A four step purification procedure for polyclonal human serum IgM was elaborated, including ultracentrifugation, ammonium sulfate and polyethylenglycol precipitations and diffusion-exclusion gel chromatography. IgM was glycated in vitro both in the presence of [C-14]glucose and with unlabeled glucose. Influence of incubation time up to 10 days and of glucose concentration between 10 and 60 mmol/l were studied. With 10 mmol/l glucose, a molar ratio glucose/IgM of 5.7 was attained in 10 days. Increase of glucose concentration up to 60 mmol/l led to a molar ratio of 16.0. Both basal and in vitro glycation were evaluated by H-3-labeling by gel filtration and Concanavalin A-Sepharose chromatographies. Glycation occurs mainly on the heavy chains (>85%), particularly on the Fd region.