Capillary zone electrophoresis for the study of the binding of antithrombin to low-affinity heparin.

When low-affinity interactions between glycosaminoglycans and precious proteins are studied, it is imperative to design an experimental set-up that consumes as little material as possible. To evaluate the applicability of the CZE technique to this problem, we explored the interaction between antithrombin and low-affinity heparin. In a series of CZE experiments we demonstrated that the mobility of antithrombin increases gradually as increased concentrations of low-affinity heparin were added to the electrolyte. The results were, as expected, consistent with the general algorithm for monovalent binding. The binding constant was estimated at 20+/-6 microM in excellent agreement with the value reported in the literature.

Fast, high-resolution (capillary) electrophoresis in buffers designed for high field strengths.

Capillary electrophoresis in conventional buffers and in 50 microns capillaries permits field strengths as high as 300-500 V/cm with acceptably low thermal zone deformation. However, still higher field strengths (up to at least 2000 V/cm) can be applied without a decrease in resolution if the experiments are performed in the buffers described in this paper. Characteristic of these buffers is their low electrical conductivity and yet satisfactory buffering capacity accomplished either (i) by selecting buffer constituents of relatively high molecular weight and small net charge or (ii) by fractionation of carrier ampholytes (originally introduced for isoelectric focusing experiments) into a series of narrow pH range fractions and using these fractions as buffers, or (iii) by selecting an ampholyte with two acidic groups and one basic group (or one acidic group and two basic groups) and with a pI value close to two of its pK values. In such buffers, aromatic carboxylic acids and proteins used as model substances could be analyzed rapidly. For instance, albumin and transferrin were separated at 30,000 V (1.99 microA) in 15 cm long fused silica capillaries (50 microns ID) within 40 s and the carboxylic acids within 25 s. The resolution was similar to that obtained at standard voltage (5000 V; 0.33 microA), but the analysis time was reduced sixfold. Although not verified experimentally we also suggest the use of relatively high-molecular-weight polyoxyethylene derivatized with one acidic group (for instance, boric acid) and one basic group (an amine), both having the same pK value, which should afford both a very high buffering capacity and very low electrical conductivity (at low buffer concentrations).