ISOLATION, CHARACTERIZATION AND RADIOIMMUNOASSAY OF RAT ALPHA-MACROFETOPROTEIN (ACUTE PHASE-A2 MACROGLOBULIN)

Rat alpha-macrofetoprotein (AMF)§ has been isolated and purified by antibody affinity chromatography and quantitated using a double antibody radioimmunoassay (RIA) with a sensitivity of 20 ng. Two forms of AMF, differing in reference mobilities (R) in 5% polyacrylamide gels and in isoelectric focusing points (pI), are present in serum. The Rf0.10 AMF has a pI of 4.35 and Rf0.25 AMF a pI of 4.65. Rf0.25 AMF competes 2.2 times better than Rf0.10 AMF in an RIA with 125I Rf0.25 AMF as the predominant antigen. The two forms do not differ substantially in mol. wt. AMF has a mol. wt of 700,000 ± 70,000 determined by SDS polyacrylamide gel electrophoresis. Dithiothreitol reduction of AMF resulted in six different chains, the largest with mol. wt 175,000. It is suggested that the five smaller chains are the result of interaction of AMF with endoproteases. The E1% 280 of AMF is 9.16 ± 0.3 by the biuret method using bovine serum albumin as a standard and 10.6 ± 0.3 by microKjeldahl N analysis. Amino-acid analysis of AMF is compared with the analyses recently reported by others. Normal adult rat serum AMF concentrations determined for two groups of rats are 17 ± 2 and 32 ± 6 μg/ml. © 1979.