STUDIES ON CHYMOTRYPSIN C AND PAPAIN FRAGMENTS OF HUMAN IMMUNOGLOBULIN M

Human Waldenstrom macroglobulins and their reductive subunits were subjected to proteolysis by chymotrypsin C (isolated from porcine pancreas) and papain. Chymotrypsin C cleaved immunoglobulin M into three fragments which had sedimentation coefficients of Cl = 16.6 S, CII = 6.6 S, and CIII = 3.8 S. Antigenic analysis showed that CIII with the molecular weight of 40,000 corresponds to an Fabμ fragment. CII with the molecular weight of 135,000 is a dimer of CIII plus an additional fragment of 55,000. Cl is a mixture which can be resolved by equilibrium ultracentrifugal technique into two major components with weight-average molecular weights of 773,000 and 606,000, and is composed of immunoglobulin M molecules from which one or more F(ab′)2μ fragments have been hydrolyzed. The calculation of the yield of Fabμ from proteolysis of the reductive subunit demonstrated the presence of 2 Fabμ units in the 7S subunits and 10 in the parent 19S immunoglobulin M. Short-term digestion of immunoglobulin M by papain produced three fragments which were separable on Sephadex gel filtration and had sedimentation coefficients of PI = 18.4 S, PII = 7.0 S, and PIII = 3.7S. PIII contains two components corresponding to Fabμ and Fcμ fragments. After longer periods of incubation with papain, PHI (Fabμ fragments) with the molecular weight of 37,000 are the only immunologically reactive fragments detectable and the remainder of the molecule is degraded into peptides. It is believed that in the presence of 0.01 m cysteine immunoglobulin M is reduced to the reductive subunit of immunoglobulin M (PII) and then the 7S subunit is degraded into PHI by papain. PI is most likely the unreduced and undigested immunoglobulin M. Quantitative complement fixation techniques have been used to study the immunological relationships between the proteolytic fragments and to localize the antigenic determinants of various antisera including those responsible for the individual (idiotypic) specificity. Nearly one-third of the total hexose found in the reductive subunit of immunoglobulin is bound to an F(ab′)2μ fragment. Hence, over two-thirds of the carbohydrate resides in the C-terminal region beyond the chymotrypsin C cleavage. The results of these studies are consistent with a circular pentameric model of immunoglobulin M containing 10 Fabμ units. © 1969, American Chemical Society. All rights reserved.