An apparatus for membrane-confined analytical electrophoresis.

A membrane-confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377-382). Presented here is a design for this apparatus, which permits the on-line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady-state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused-silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 microL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.

Insights from a new analytical electrophoresis apparatus.

Charge is a fundamental property of macromolecules that is inextricably linked to their structure, solubility, stability, and interactions. Progress has been made on the theoretical and structural aspects of these relationships. However, for several reasons, charge is difficult to measure in solution. Consequently, there is a lack of experimental data that, independent of other macro-ion properties, determines the effective charge. To overcome this problem, novel instrumentation and methods are being developed in our laboratory. Described here is an analytical electrophoresis apparatus that permits both the measurement of electrophoretic mobilities and the determination of steady-state electrophoresis concentration distributions. The latter provides a different-perspective on the processes that influence macro-ion behavior in an electric field. In addition, the apparatus permits the determination of diffusion coefficients either from boundary spreading during transport or from the decay of a concentration gradient. All of these determinations can be made with a single, 8-microL sample in a variety of solvents, thus providing unique insights into the charge properties of a macro-ion. Presented here is a progress report about this emerging technology, including the description of a prototype apparatus and examples of its use with a DNA oligonucleotide.

Direct determination of macromolecular charge by equilibrium electrophoresis.

Charge is a fundamental property of macromolecules in solution. However, estimation of the apparent charge on polyions has confounded science for decades. Presented here is a general method to determine directly the apparent charge on a polyion, regardless of its size or shape. This new method uses equilibrium electrophoresis, a procedure in which opposing solute flows from electrophoresis and from diffusion balance everywhere as the system reaches a steady-state distribution. The method uses only small quantities of materials, is nondestructive, and requires only simple, inexpensive instrumentation. Here we describe a prototype apparatus, demonstrate the phenomenon, and present experimental examples of the procedure.

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation.

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.