Selective purification of sex-influenced esterase from rat serum by immunoaffinity chromatographies.
Sex-influenced esterase (ES-SI) is a female-specific serum esterase of rats. There are two alleles at the Es-Si locus, Es-Sia coding for ES-SI and Es-Sib, a silent allele. ES-SI was purified to electrophoretic homogeneity from the serum of adult female SI3 rats by a four-step purification. First, albumin was removed from the serum by Blue-Sepharose CL-6B affinity chromatography. Sera of rats carrying Es-Sia and Es-Sib were utilized to make two novel immunoaffinity columns. Rat anti-(ES-SI) alloantibody was obtained after the immunization of rats carrying Es-Sib with rat ES-SI-positive serum. ES-SI bound to the gel in this antibody-conjugated affinity chromatography. Simultaneously, rabbit anti-(rat serum protein lacking ES-SI) antibody was raised against ES-SI-negative rat serum and used to exclude other serum proteins. ES-SI was recovered in the flow-through fractions in this antibody-conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES-SI was selectively purified about 240-fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES-SI was purified by preparative polyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/polyacrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES-SI was 30-fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES-SI was selectively purified 2280-fold comparing its recovery to that of total protein. ES-SI exhibited a pI of 4.6 and optimum pH of 6.7. The activity of ES-SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.
Hirose
T
T
Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Morita
A
A
Yamada
M
M
Nikaido
H
H
Hayakawa
J
J
Nikaido
O
O
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007106:Immune Sera; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011921:Rats, Inbred WKY
D007106:Immune Sera; D002265:Carboxylic Ester Hydrolases