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Circular dichroism determination of class I MHC-peptide equilibrium dissociation constants
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Chantal S. Morgan James M. Holton Barry D. Olafson Pamela J. Bjorkman Stephen L. Mayo 《Protein science : a publication of the Protein Society》1997,6(8):1771-1773
Class I major histocompatibility complex (MHC) molecules bind peptides derived from degraded proteins for display to T cells of the immune system. Peptides bind to MHC proteins with varying affinities, depending upon their sequence and length. We demonstrate that the thermal stability of the MHC-peptide complex depends directly on peptide binding affinity. We use this correlation to develop a convenient method to determine peptide dissociation constants by measuring MHC-peptide complex stability using thermal denaturation profiles monitored by circular dichroism. 相似文献
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The dissociation constants of tetrahydrofolic acid 总被引:2,自引:0,他引:2
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The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals 总被引:25,自引:0,他引:25
Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method. 相似文献
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It is commonly believed that the tetrameric Adair constants for oxygen binding to human hemoglobin can be evaluated from a single oxygenation experiment at 'high' hemoglobin concentration without considering the consequence of the presence of alpha beta dimers. We present examples which demonstrate that this is a very dangerous assumption. Without a knowledge of the complete oxygenation-linked dimer-tetramer association reaction (alpha beta Xi----(alpha beta)2Xj), it is impossible to predict a priori how high of a hemoglobin concentration would be required to make this assumption. Furthermore, without a knowledge of the complete oxygenation-linked dimer-tetramer association reaction, it is impossible to predict a priori the direction and magnitude of the systematic errors which are induced by making this assumption. 相似文献
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The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin-biotin. The distribution coefficient calculated from (Ve-V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes. 相似文献
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Winzor DJ 《Analytical biochemistry》2011,(2):273-277
Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants (Kd) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium–ethylenediaminetetraacetic acid (Cd–EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen–antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads—a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based. 相似文献
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Hiroshi Watari Yoshinori Isogai Hiroyasu Nlshikawa 《Journal of theoretical biology》1980,83(2):209-214
The maximum slope of the plot, appearing in the paper of Watari & Isogai (1976), was derived algebraically as a function of allosteric constants c and αmor βm (= cαm), and the relation between L, c, and αmor βm, was also obtained, where are concentrations of unligated R and T states respectively, KRand KT are microscopic dissociation constants, and Fm is the ligand concentration at the maximum slope of the plot. When the maximum slope is increased by one, the value becomes Hill constant, n. Nomographs which enable easier estimation of allosteric constants, L and c, were constructed from the two given values, the maximum slope of the plot, n ? 1, and αmor βm, in the cases where the maximum number of ligands, N, was 2 and 4. In the nomograph, log c is plotted against log L2cN keeping the value of the maximum slope of the plot and that of αmor βm constant. These nomographs show that the representation is symmetrical in the cases of L2cN > 1 and L2cN < 1. 相似文献
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The application of copolymerized agarose-polyacrylamide gels as the support for immobilized Cibacron Blue F3G A is demonstrated for the analytical electrophoresis of proteins possessing an affinity for this dye. Bovine serum albumin was used as a model protein to develop this technique. The optimal conditions for preparing matrices are described. These conditions produce gels with suitable mechanical strength and which allow rapid electrophoresis of proteins. The dye-agarose-polyacrylamide gels permit the determination of dissociation constants. The ease of preparation of these matrices recommends them for a variety of quantitative analytical investigations. 相似文献
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The dissociation of carbon monoxide from hemoglobin intermediate 总被引:1,自引:0,他引:1
M Samaja E Rovida M Niggeler M Perrella L Rossi-Bernardi 《The Journal of biological chemistry》1987,262(10):4528-4533
To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species. 相似文献
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Observation of the dissociation of unliganded hemoglobin 总被引:9,自引:0,他引:9
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S G Waley 《The Biochemical journal》1982,205(3):631-633
The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants. 相似文献