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Biomedical subjects

I Pecht

Publications and source records attributed to I Pecht.

At least 181 records · Page 10Linked to original sources
PubMed1978

Binding site of a dextran-specific homogeneous IgM: thermodynamic and spectroscopic mapping by dansylated oligosaccharides.

The hapten binding properties of the homogeneous mouse IgM secreted by MOPC-104E were investigated. Hapten-association constants were determined either by equilibrium and displacement equilibrium dialysis or by fluorometric titrations of the protein with the fluorescent derivatives of the haptens. For the latter type of measurements, several oligosaccharides were derivatized to the corresponding dansylhydrazones. The synthesis, generally applicable to oligosaccharides with free reducing ends, is described. Analysis of the thermodynamic parameters for the binding of 18 haptens forms the basis for proposing a model of the binding site of MOPC-104E. This model is supported and refined by results of the measurements of linear and circular polarization of the fluorescence of the dansylated haptens. The binding site is proposed to consist of a cavity with about 12-A depth, complementary to a terminal nonreducing nigerosyl group. At the entrance to this cavity, a further subsite is identified forming interactions of lower specificity with an additional glucose unit.

Binding Sites↗
PubMed1978

Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation.

The interaction of oligogalactan haptens with the murine myeloma proteins XRPC-24 and J-539 has been investigated by the fluorescence temperature-jump method. The relaxation spectrum is composed of two processes, the faster representing hapten assocaition and the slower a protein isomerization. In both cases the concentration dependence of relaxation times and amplitudes was consistent with the general mechanism formulated by Lancet and Pecht (1976, Proc. Natl. Acad. Sci. U.S.A. 73:3549), in which the equilibrium between two conformations of the protein is shifted by hapten binding. The intact proteins and their Fab fragment had identical kinetic behavior, indicating that the conformational changes are located in the Fab region. Temperature dependence analysis for protein J-539 permitted the calculation of activation parameters and led to a consistent energy profile for all the elementary steps. The conformational states are separated by large activation barriers, but have similar free energies. The results suggest that hapten-induced conformational changes in immunoglobulins are more general phenomena than was previously thought.

Animals↗
PubMed1978

Reductant-dependent electron distribution among redox sites of laccase.

Rhus laccase (monophenol monooxygenase, monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) an O2/H2O oxidoreductase containing four copper ions bound to three redox sites (type 1, type 2, and type 3 Cu pair), was titrated anaerobically with several reductants having various chemical and thermodynamic properties. The distribution of electron equivalents among the redox sites was found to be reductant dependent. When the data for titration by various reductants of the type 3 site were plotted against those of the type 1 site according to the Nernst formalism, the slope n varied from 2.0 to 1.0. The redox potential of the reductant's first oxidation step is qualitatively correlated with the value of n and is suggested as the factor that modulates the electron distribution. Such a behavior implies a nonequilibrium situation. A very good simulation of the data was provided by an analysis assuming a formally variable cooperativity between the two type 3 copper ions. This apparent variability is suggested to result from a process whereby sufficiently strong reductants induce a transition of the type 3 site from a cooperative two-electron acceptor to a pair of independent one-electron acceptors. This uncoupled state of the type 3 site is considered metastable. Other possible models were also investigated. Summarizing the available data, we conclude that the two-electron accepting behavior of the 330-nm chromophore is the exception rather than the rule.

Anaerobiosis↗
PubMed1977

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.

Antibody Specificity↗
PubMed1976

Kinetics and equilibria of the electron transfer between azurin and the hexacyanoiron (II/III) couple.

The electron transfer reaction between the "blue" single copper protein azurin (from Pseudomonas aeruginosa) and the hexacyanoiron (II/III) couple has been studied. Equilibrium constants for the reduction of azurin were measured spectrophotometrically in the temperature range 5-33 degrees C (K = 1.1 X 10(-2) at 25 degrees C, deltaH degrees = 10.9 kcal/mol, 0.1 M potassium phosphate, pH 7.0, I = 0.22). The enthalpy change was also determined by microcalorimetry and from the analysis of chemical relaxation amplitudes. Following a temperature-jump perturbation of this equilibrium, only a single relaxation was observed. The reciprocal of the relaxation time increased linearly as oxidized azurin was reacted with increasing amounts of ferrocyanide, yet reached saturation when reduced azurin was titrated with ferricyanide. This behavior as well as the analysis of the relaxation amplitudes led to the following scheme for this system: see article. At 25 degrees C the rate constants for the electron transfer were k+3=6.4s-1 and k-3=45s-1, the association constants K1=54 M-1 and K2-1=610 M-1. The activation and overall thermodynamic parameters as well as the individual thermodynamic values for the different steps were combined to construct a self-consistent energy profile for the reaction.

Azurin↗