Studies on antithrombin III. Purfication of canine antithrombin III and its properties

Abstract

Canine antithrombin III was purified by the use of heparin-agarose affinity chromatography according to the method described by Damus and Wallace. Since the AT III prepared by this method contained a trace amount of impurity, we further purified it by preparative polyacrylamide gel electrophoresis. The protein thus obtained showed a single band on polyacrylamide gel electrophoresis. Antiserum against this protein produced in rabbits made a single precipitation line with canine plasma. It showed both antithrombin activity and heparin cofactor activity and inhibited thrombin activity progressively. From these data, we concluded that our AT III was pure so far as we could determine. The molecular weight of this AT III was 70, 000, when measured by the method of Andrews using Sephadex G 200 column chromatography. The extinction coefficient was determind by measuring the weight of well-dried lyophilized materials dialysed against distilled water extensively and measuring the optical density of them dissolved in 0.05M. phosphate buffer (pH 8.0) at 280nm using 1cm quartz cuvette. The extinction coefficient of AT III was 6.2. The specific activity of purified AT III was 400 units per mg, when the activity of 1ml of defibrinated plasma was set at 100 units arbitrarily.Canine AT III metabolism was studied in 4 dogs of both sexes using I-125-AT III as a tracer. AT III was labeled by the method of McFarlane. The average plasma behavior of injected I-125-AT III could be closely expressed by the two exponential function of the form following, x=0.53e-0.408t+0.47e-3.01t. The plasma half life of I-125-AT III averaged 1.7±0.2 days. The calculated tracer data were; the amount of plasma AT III (x) was 32.0±3.3mg/kg, the extravascular AT III (y) was 16.6±2.6mg/kg, fractional transcapillary transfer rate to extravascular space (j1) was 0.94±0.19/day, the fractional return rate of y to plasma (j2) was 1.81±0.29/day, fractional catabolic rate (j3) was 0.71±0.10/day. The transcapillary flux (j1x) was 30.0±4.7mg/kg/ day and catabolic flux (j3x) was 22.3+1.8mg/kg/day.These data were compared with the data which were previously obtained using AT III preparation purified by another purification method described by Rosenberg et al, and the dfferences between them were discussed.