The determination of equilibrium constants for heterogeneous macromolecular interactions. Systems forming 2:1 complexes.

In developing a method for analyzing the heterogeneous association nA + mB in equilibrium AnBm, we have specifically investigated the case of n = 2, m = 1 for both the specific case of no appreciable intermediates and the more general case allowing intermediates. Computer-simulated three-dimensional surfaces of the 2:1 model generated from total concentrations of species A and B and the resulting weight-average molecular weights were analyzed with a Gauss-Newton nonlinear least-squares minimization routine. The surfaces generated included normalized random error of varying standard deviations imposed upon both the concentrations and weight-average molecular weights. For comparison purposes, these surfaces were analyzed not only by using the correct 2:1 model, but also by an incorrect (1:1) model and by the other (incorrect) 2:1 model. Except for those situations where the 'experimental' noise was consistently higher than the concentration of one of the species, correct K values were obtained and the correct model was easily distinguished from the incorrect model. The computer routine similarly distinguished between data correctly described as 1:1 and the same data incorrectly analyzed as either 2:1 model. For those cases in which a microscopic Ki value predicts an association such that all species involved for that particular Ki are in appreciable amounts, the Ki value is returned correctly. Correct overall equilibrium constants are also converged upon as long as adequate amounts of A2B, B and A are present.

The determination of equilibrium constants for heterogeneous macromolecular interactions.

A method has been developed to determine the association constant for a heterogeneous association of the type A + B in equilibrium AB. This method requires knowledge of the two initial concentrations and of the resulting weight-average molecular weight for each data point. Computer simulations using Gaussian-distributed error on the measured parameters show that the researcher can readily determine whether the particular concentration range chosen is appropriate for the strength of binding and therefore how reliable the calculated constant might be. It is also shown that errors in measuring molecular weight have, in general, a more profound effect than do errors in concentration.

Stepwise sequence determination from the carboxyl terminus of peptides.

The thiocyanate method for stepwise degradation of peptides from their COOH termini [Stark, G. R. (1968) Biochemistry 7, 1796] has been investigated. The method involves first the reaction of the COOH-terminal residue with thiocyanate in an activation solvent of acetic acid and acetic anhydride and then cleavage of the COOH-terminal residue as its 2-thiohydantoin by acetohydroxamate in aqueous solution. The two steps of the degradation have been studied by using model peptides, and conditions have been developed for the rapid efficient removal and identification of the COOH-terminal residue of short peptides. The methods have been applied to peptides that have been covalently attached to insoluble supports. In this solid phase version of the degradation, a highly substituted porous glass activated with N,N'-carbonyldiimidazole has been prepared for use as the insoluble support. A number of peptides have been coupled to the porous glass, and several rounds of the degradation have been performed on immobilized peptides. High-pressure liquid chromatography provides a rapid, sensitive identification method for the 2-thiohydantoins. In addition, gas-liquid chromatography of the amino acid 2-thiohydantoins and reconversion to the parent amino acid have been used to identify the cleaved residues. The method of sequential degradation has been applied to a number of short model peptides such as Gly-Leu-Tyr, Met-enkephalin, and Val-Leu-Ser-Glu-Gly and has been used to determine the COOH-terminal sequence of 4 residues of a 22-residue cyanogen bromide fragment of pygmy sperm whale myoglobin.