Binding of the Triton X series of nonionic surfactants to bovine serum albumin.

The binding of a series of Triton X nonionic surfactants (NIS) tobivine serum albumin (BSA) has been studied by equilibrium dialysis and titration calorimetry. At pH 7.0, Triton X molecules bind to two classes of sites, the first 2 molecules binding with positive cooperativity to high-affinity sites following by the binding of approximately 15 additional molecules to lower affinity, thermodynamically identical, and independent sites. The strength of the binding decreases as the number of oxyethylene units is increased in the surfactants Triton X-114, X-100, X-102, and X-165. Calorimetric measurements show the enthalpy change for the NIS-BSA interaction to be small and endothermic. Increasing the hydrophilic oxyethylene chain length results in a more endothermic enthalpy change and a smaller association constant. Electron spin resonance studies of Triton X binding to BSA, covalently spin-labeled with N-(2,2,6,6-tetramethyl-piperidinyl-1-oxy)maleimide, indicated that the protein conformation in the vicinity of the labeled sulfhydryl was insensitive to NIS binding from dilute monomeric solutions. Calorimetric experiments near the critical micelle concentration indicate, however, that the protein probably undergoes a conformational change associated with the population of the lower affinity NIS binding sites.

Distribution of transplantation antigens on cell surfaces.

Significant advances have been made over the past few years in elucidating the genetics, the chemical composition, and, more recently, the in situ relation of the major histocompatibility antigens of the mouse and man. Attempts to map the arrangement of individual antigens on the surface of cells have revealed that some antigens specified by a given subregion of both the H-2 and HL-A systems are in close proximity on the cell membrane and that attachment of antibody to one site to a certain degree blocks or inhibits the binding of antibody to the adjacent site. Allelic antigens in the H-2 system tend to inhibit binding. H-2D and H-2K antigens show either inhibition or noninteraction, possibly reflecting a cis-trans effect. Unlike with the H-2, inhibition of binding occurs only between HL-A antigens specified by homologous chromosomes. Also, a number of instances have been noted where inhibition of binding is unidirectional, possibly reflecting a polymeric nature of antigen or stratification of moieties at cell surface. Inhibition of antibody attachment between several alloantigenic systems on thymocytes in mice and also a variation in the mobility of the histocompatibility antigens suggest that the moieties bearing histocompatibility antigens are comprised of several gene products. Further work is needed to establish the validity of this assumption and to fully define the composition of these units. Ample data have been obtained from both biological and biophysical experiments to support the suggestion that single or multiple complexes of glycoproteins can move in the plane of the membrane. Although the composition of these cell membrane components remains a question, direct visualization by fluorescence and electron microscopy indicates that these moieties are small and, under natural conditions, distributed uniformly over the cell surface. Direct and indirect labeling techniques have shown that the complexes have no fixed position in the cell membrane and can be displaced laterally in the plane of the membrane without affecting the distribution of other surface molecules, such as sIg and species-specific antigens. Additional evidence suggests that H-2D and H-2K antigen complexes, as well as their gene products specified by different parent chromosomes, may be displaced separately. These observations are especially interesting and must be reconciled and data obtained by proximity analysis which indicate an association of some allelic products and possibly certain combinations of D and K antigens. Whether the differences noted in reactivity of the various surface antigens following attachment of antibodies are attributable to difference in size or to differences in the manner of their intercalation in the cell membrane remains to be elucidated. The rapid advances in elucidation of the molecular structure of biological membranes suggest that experimental work should be done on the biophysics of the structure of the antigenic sites and the mechanism of migration...

An EPR study of structural perturbations induced by methylindole in the protein and lipid regions of erythrocyte membranes.

3-Methylindole has been shown in previous work to cause pulmonary edema and emphysema in cattle and goats. In this paper, evidence is presented to show that 3-methylindole induces structural perturbations in bovine erythrocyte membranes. The structural perturbations which were induced as a function of 3-methylindole concentration in the membranes were measured by EPR using the attachment of maleimide spin label to the sulfhydryl groups of membrane proteins and by intercalation of methyl-5- doxylstearate, methyl-12-doxylstearate, and methyl-16-doxylstearate into the lipid region. The EPR spectra of the malemide spin-labeled membrane proteins became more immobilized as the concentration of 3-methyl-indole increased. The order parameter describing the EPR spectra of methyl-5-doxylstearate decreased from 0.69 to 0.55 as the concentration of 3-methylindole increased. The acyl chains in the region of the carbon 5 position were converted to a less ordered structure. The EPR-spectra of methyl-12-doxylstearate was a superposition representing at least three tumbling rates. As the concentration of 3-methylindole increased, the major fraction of the methyl-12-doxylstearate probes experienced an increase in tumbling rate and a smaller fraction is observed a strongly immobilized state. The EPR spectra of methyl-16-doxylstearate were not perceptibly changed in the presence of 3-methylindole. The concentration dependence suggests that 3-methylindole preferentially intercalates into the ordered region of the alkyl chains sampled by the methyl-5-doxylstearate. These results confirm that 3-methylindole induced structural changes at the molecular level.

Spectral studies of iron coordination in hemeprotein complexes: difference spectroscopy below 250 millimicrons.

In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mmu, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mmu, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mmu is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented.