Determination of protein association constants by electrophoresis.

An electrophoresis cell with scanning UV-absorption optics is presented. It allows the measurement of moving reaction boundaries of dilute protein solutions with a high-resolution. The protein profiles in the boundaries can be extrapolated to infinite time after an appropriate transformation of space and time coordinates and then evaluated with respect to association constants. This is demonstrated for the dimer-tetramer equilibrium of haemoglobin.

An osmometer for aqueous biopolymer solutions.

A sensitive osmometer for the study of the osmotic pressure of aqueous protein solutions is presented. Equilibrium is reached within 1 to 2 h and is reproducible to about 1 N.m-2 (= 0.01 cm H2O). The method allows the determination of dissociation constants of proteins down to 1 x 10(-8) M.

Osmotic pressure measurements of the ligand-linked dissociation equilibrium of human oxyhemoglobin.

The tetramer/dimer equilibrium of human oxyhemoglobin has been measured by an osmometric method as a function of pH, chloride and 2,3-bisphosphoglycerate concentrations at 21 degrees C. The binding constants of chloride and 2,3-bisphosphoglycerate to the tetramer and the dimer were thus evaluated at various pH values. 2,3-Bisphosphoglycerate is bound to the dimer more strongly than to the tetramer. The dimer has a second binding site for 2,3-bisphosphoglycerate. The data for 2,3-bisphosphoglycerate binding were corroborated by direct binding measurements with the ultrafiltration method.

Measurement of rapid membrane permeation in cell suspensions by application of a generalized capillary method.

An improved version of the capillary technique for the determination of diffusion coefficients has been developed as a simple method of measuring membrane permeabilities of single cells suspended at relative densities between 0.70 and 0.97. A new, generalized theoretical formulation to describe the diffusion process of a solute in a composite system was derived using a series-parallel-pathway model with explicit consideration of the diffusion pathways inside and between the cells. This renders the technique insensitive to unstirred layer effects. Any single cell population of known size distribution may be investigated. High permeabilities (above 5.10(-3) cm/s) can be measured with the greatest precision, but lower permeabilities, down to a limit of about 5.10(-4) cm/s, may also be determined by the method. Measurements in erythrocyte suspensions have been made using non-electrolytes such as hexanol, water and ethylene glycol as test solutes. The permeabilities obtained agree with the values obtained by much more sophisticated equipment. Cell shape was shown to be without significant influence on the permeability data obtained. The procedure may become of particular interest for measurement of suspensions of membrane vesicles.

Identification of different quaternary structures of beef heart cytochrome-c oxidase by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional gel electrophoresis is described to identify different quaternary structures of the heart cytochrome-c oxidase. Bovine enzyme was purified and separated by discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the 1st dimension into several discrete complexes and thereupon shown to be heterodisperse in Triton X-100 and dodecyl maltoside. A discontinuous SDS-polyacrylamide gel electrophoresis in the 2nd dimension was used to determine the subunit composition of the isolated complexes. One of these represents the intact enzyme with 12 different polypeptides while the others have an incomplete subunit composition.

A simple technique of measuring high membrane permeabilities of human erythrocytes.

A new way of measuring high diffusional membrane permeabilities of intact erythrocytes is presented using THO and 14C-glycol as test solutes. The technique combines the theoretical approach used by Redwood, Rall and Perl (J. Gen. Physiol. 64:706-729, 1974) and an experimental procedure introduced by Wang (J. Am. Chem. Soc. 73:510-513, 1951), which greatly simplifies the performance of the experiments. Permeability coefficients obtained by the new technique compare well to data derived by the approaches hitherto available. In view of its simplicity our method may be appropriate for the serial experiments necessary to characterize the transport mechanisms of water and other highly permeable lipophilic nonelectrolytes and for studies on other single cell systems.

On the determination of association constants of proteins by electrophoresis measurements.

During the electrophoresis of a reversibly associating substance the concentration profile is determined by diffusion, migration and reaction. The influence of the diffusion can be eliminated by extrapolating the concentration profiles, taken at different times and suitably transformed, to infinite time. This leads to a profile which reflects migration and reaction only (Gilbert profile). From this the association constant can be deduced. Preliminary experiments with beta-lactoglobulin A show the feasibility of the method.

Approximate solution of the differential equation for the migration of a reversibly reacting substance.

A series solution is given for the differential equation describing the transport of a reversibly reacting substance in an infinite rectangular cell. The concentration dependence of the constituent transport coefficients has been approximated by polynomials. The solution converges for short time (t is less than 1 h). Several features of the leading and trailing boundary of monomer-trimer and monomer-dimer-trimer systems are discussed.