Determination Of Antithrombin III Heparin-Binding Capacity By High Performance Liquid Chromatography

The heparin-binding capacity of purified human antithrombin III (AT) both native and after a variety of denaturat- ing conditions was measured using a high performance liquid chromatographic (HPLC) system.

An exclusion chromatography, TSK-3000, 0.75 (ID) × 50 cm column equilibrated with 0.1M NaCl in 0.02M potassium phosphate buffer pH 7.35, at a flow rate of 1 ml/min was used. Under these conditions two essentially resolved peaks were obtained at 10.5 to 14 min. The peak corresponding to the unbound AT, or native AT in the absence of heparin (H), is sharp; that of the AT-H complex is broad due, probably, to the size heterogeneity of the H used in the tests. Without H, aggregates of AT are also resolved by this technique. The areas under the curve of the protein peaks monitored spectrophotometrically in the absence and in the presence of an excess of H are used to quantitate these species. A titration of AT with increasing amounts of H indicates the concentration of H sufficient to bind all the H-binding AT present in a given AT containing sample.

The results of the HPLC method correlate well with those obtained by two-dimensional or crossed Immunoelectrophoresis (CIEP), a technique that can be used to estimate the H-binding capacity of AT. Thus, for example, in non- pasteurized, pasteurized and heated AT samples, for which the HPLC method indicated that 5, 25 and 80% of the AT had lost its H-binding capacity, the CIEP gave similar results.

The results indicate that the HPLC method can adventage- ously be used to determine quantitatively the H-binding capacity of AT and, if present, AT aggregates, thus appearing more informative than CIEP. The analysis time is less than 30 minutes and uses inexpensive reagents.