An improved method for removal of antithrombin III from post-heparin plasma lipase fractions with a high recovery rate.
Affinity chromatography on heparin-Sepharose has been widely used for the purification of post-heparin plasma triglyceride lipases, but this procedure alone yields lipase fractions with a high content of antithrombin III (AT), which also binds to heparin and coelutes with the lipases. A rapid procedure in which this major contaminant is reduced by the use of affinity chromatography on heparin-Sepharose with low affinity for AT and further removal by anti-AT IgG Affi-Gel 10 column chromatography is reported and the results are compared with these of the conventional heparin-Sepharose and Concanavalin A-Sepharose column chromatography two step method. While the AT contamination of hepatic lipase eluate from conventional heparin-Sepharose column chromatography was 92 mg/dl in 100 mg/dl protein, heparin-Sepharose with low affinity for AT yielded samples with 55 mg/dl in 100 mg/dl protein. A second passage on concanavalin A-Sepharose of the sample from conventional heparin-Sepharose reduced the AT content from 92 mg/dl to 42 mg/dl in 100 mg/dl protein, but passage of the sample from heparin-Sepharose with low affinity for AT through an anti-AT IgG Affi-Gel 10 column removed almost all of the AT contamination from the lipase sample. This hepatic triglyceride lipase (HTGL) sample had a specific activity 1,963-fold higher than that of post-heparin plasma with a yield of 17% of the starting material, showing an excellent recovery rate compared with similar methods in use.
Moriguchi
E H
EH
Department of Internal Medicine, Tokai University School of Medicine, Kanagawa, Japan.
Tamachi
H
H
Homma
Y
Y
Goto
Y
Y
D000990:Antithrombin III / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008049:Lipase / Q000097:blood*; D012685:Sepharose / Q000031:analogs & derivatives
C037405:heparin-sepharose; D000990:Antithrombin III; D012685:Sepharose; D008049:Lipase