Calculation of the Spectrum of Chemical Relaxation Times for a General Reaction Mechanism

Castellan, Gilbert W.

doi:10.1002/bbpc.19630670911

Cited by 103 publications

(24 citation statements)

References 11 publications

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“…KT/KR = K0/K, [4] A maximum of three relaxation times may therefore be observed, and when the isomerizations are much slower than the associations (as borne out by experiment) one slow monomolecular time and two fast bimolecular times are expected (14).…”

Section: Results and Interpretationmentioning

confidence: 95%

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Kinetic evidence for hapten-induced conformational transition in immunoglobin MOPC 460.

Lancet¹,

Pecht²

1976

Proc. Natl. Acad. Sci. U.S.A.

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The kinetics of hapten binding to the homogeneous immunoglobulin A secreted by the murine plasmacytoma MOPC 460 was investigated by the chemical relaxation method. Two distinct relaxation times were observed in the binding equilibrium with three different haptens. A detailed concentration dependence analysis of relaxation times and amplitudes was performed with the hapten epsilon-N(2,4-dinitrophenyl)-lysine (Dnp-Lys). The results support a mechanism in which two interconvertible conformational states of the protein bind the hapten with different association constants. Hapten binding shifts the equilibrium towards the better binding state. These observations form kinetic evidence for a conformational transition induced in the immunoglobulin by ligand binding to its antigen binding site, and are in line with the allosteric hypothesis for the initiation of physiological functions by antigen-antibody association.

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Section: Results and Interpretationmentioning

confidence: 95%

“…12) also through the quenching of the hapten fluorescence as well as changes in its absorption. (14) and Jovin (15), respectively. The experimental concentration dependences were fitted to those predicted by different mechanisms using the algorithm of Powell (16).…”

mentioning

confidence: 99%

Kinetic evidence for hapten-induced conformational transition in immunoglobin MOPC 460.

Lancet¹,

Pecht²

1976

Proc. Natl. Acad. Sci. U.S.A.

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“…The relationship of the affinity to the reaction advancement for each step and the coupling between equilibria is expressed in the diagonal and off-diagonal terms, respectively, of the g matrix as shown by Castellan [31]. For the scheme of Eqn (2) it is given by: (see p. 344).…”

Section: Appendixmentioning

confidence: 99%

Electron Transfer between Azurin from Alcaligenes faecalis and Cytochrome c551 from Pseudomonas aeruginosa

1981

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The electron transfer equilibrium and kinetics between azurin from Alruli~enr.s,fueru/i.s and cytochrome r~s I from Pseudomonas aeruginosa have been studied. The equilibrium constant. [Az(IIj]) = 0.5 at 25°C is about seven times smaller than that observed between the cytochrome c5s1 and the azurin both from P. ae~ugizzosa [Rosen, P. and Pecht,1. ( 1 976 The kinetics of the reaction between Akaligenc~s azurin and Pseudomonas r~torlz~oznc cSs1 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction betwecn P. acwginosu azurin and cytochrome cSs1 is not observed with the AIc~ligezz~.~ azurin. The simplest mechanism that can therefore be ascribed to the investigated system is:This scheme is similar to that proposed earlier for the reaction between P. ucmginosa azurin and cytochrome ~' 5 5~ but does not involve the conformational transition proposed for azurin. The specific rates for the elcctron transfcr are still fast: 1

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“…two isomers toward ligand (L) gives rise to complex kinetics, which according to relaxation theory (10,24) should be triphasic. To our knowledge, no triphasic antibody-hapten reaction has previously been reported.…”

mentioning

confidence: 99%

“…Our stopped-flow experiments initiate from separate reactants, rather than from a point near equilibrium, hence relaxation theory (10,24) is not strictly applicable. Here we present a different treatment, which employs the approximation that the concentration of a ligand present in great excess is effectively constant over the course of a reaction, but which otherwise is exact.…”

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confidence: 99%

Conformational isomerism and the diversity of antibodies.

Foote

Milstein

1994

Proc. Natl. Acad. Sci. U.S.A.

242

215

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The fact that one cell encodes a single antibody sequence does not ne arily mean that the resulting antibody folds into a single structure, although this is a common assumpton. Here we challenge this view and suggest that many antibodies do not have a single conformation at the combining site.The basis for this proposal comes from the kinetic analysis of a set of murine hybridomas derived from defined stages of the immune response to 2-phenyl-5-oxazolone (Ox). Among them we have identified three antibodies that exhibit complex haptenbinding kinetics. We observed biphasic or triphasic reactions in stopped-flow fluorescence experiments, indicating that lignd binding involved isomerization, as well as assoiative steps. The existence of an equilibrium between at least two antibody conformations, With ligands binding preferentially to one form, was deduced from the variation with hapten concentration ofthe apparent rate of each phase.Since Clonal Selection replaced the Instructive Theory as a working model of immunity, the axiom of one-lymphocyteone-antibody has been equated to one combining site (1-4). Experiments defining the basis of humoral immune diversity have emphasized combinatorial and somatic mutational processes that lead to covalent diversity of the antibody repertoire (5). Nevertheless, two independent lines of evidence exist for diversity at the level of antibody tertiary structure. Certain crystal structures of antibodies with and without ligands have shown conformation dimorphism, which could only partly be attributed to crystallization artifacts (6-8). Despite the rich detail of these structures, crystallographically detected conformational differences due to ligation are mechanistically ambiguous (9). Differences could arise from direct interaction with the ligand (induced fit) or, with equal plausibility, by preferential ligand binding to a preexisting subpopulation of antibody isomers. This distinction is of considerable immunological consequence, since two isomers in spontaneous equilibrium would both form part of the humoral repertoire, whereas an induced conformation existing only in an immune complex would not contribute to diversity in the same way. Kinetic analysis avoids this particular ambiguity. Indeed, studies on several myeloma proteins have demonstrated kinetically distinct conformational isomers (10-13). It could be argued that myeloma cells have an uncertain ontogeny and are not subject to the immune maturation mechanisms encountered in a bona fide antigenic response; however, similar kinetic behavior was reported for an anti-fluorescein hybridoma (14). Some of these cases demonstrate a preexisting isomeric equilibrium and others are consistent with an induced-fit mechanism. Notwithstanding this evidence that conformational polymorphism can occur, the isolated examples uncovered through either structural or kinetic approaches leave an uncertainty whether antibody isomerism is an oddity or a general and important factor in an immune response.Here we address the lack of a conte...

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Calculation of the Spectrum of Chemical Relaxation Times for a General Reaction Mechanism

Cited by 103 publications

References 11 publications

Kinetic evidence for hapten-induced conformational transition in immunoglobin MOPC 460.

Kinetic evidence for hapten-induced conformational transition in immunoglobin MOPC 460.

Electron Transfer between Azurin from Alcaligenes faecalis and Cytochrome c551 from Pseudomonas aeruginosa

Conformational isomerism and the diversity of antibodies.

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