Heparin-agarose Affinity Chromatography Research Articles

Heterogeneity of the "Classical" Antithrombin III Deficiency

We investigated two thrombophilic families with the "classical" type of antithrombin III deficiency, i.e., with a low antithrombin III level measured both by immunochemical and functional methods. We obtained different antithrombin III patterns in the plasma of the affected members of the two families with the modified two dimensional immunoelectrophoresis method (heparin in agarose). In one family, the electrophoretic mobility of the antithrombin III is identical with that of normal antithrombin III. In the other, the antithrombin III displayed a decreased electrophoretic mobility in the heparinized agarose gel. The relatively low affinity of this antithrombin III to heparin could be directly proved by the heparin-agarose affinity chromatography, too. These two different antithrombin III patterns were observed by other investigators at different families as well. On the basis of our simultaneous observations of these two families we propose a classification of the inherited congenital antithrombin III deficiencies.

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

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.

Hepatic triglyceride lipase deficiency in liver disease.

The activity of post-heparin lipases in patients with alcoholic hepatitis and viral hepatitis was evaluated. Lipoprotein lipase and hepatic triglyceride lipase were differentiated by assay under high and low salt conditions and also by separation on heparin-agarose affinity chromatography columns. The mean activity of hepatic triglyceride lipase in the sera of liver disease patients was only 21-24% of the mean of controls, but lipoprotein lipase in patients' sera was not different from normal levels. Hepatic triglyceride lipase deficiency may partially account for the accumulation of a triglyceride-rich low density lipoprotein in liver disease.