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PubMed1978

Equilibrium studies of lecithin-cholesterol interactions I. Stoichiometry of lecithin-cholesterol complexes in bulk systems.

The maximum molar ratio of lecithin:cholesterol in aqueous dispersions has been reported to be 2:1, 1:1, or 1:2. The source of the desparate results has been examined in this study by analyzing (a) the phase relations in anhydrous mixtures (from which most dispersions are prepared) and (b) various methods of preparing aqueous dispersions, with the purpose of avoiding the formation of metastable states that may be responsible for the variability of the lecithin-cholesterol stoichiometry. Temperature-composition phase diagrams for anhydrous mixtures of cholesterol (CHOL) with dimyristoyl (DML) and with dipalmitoyl (DPL) lecithin were obtained by differential scanning calorimetry (DSC). Complexes form with molar ratios for lecithin:CHOL of 2:1 and 1:2; they are stable up to 70 degrees C. When x(CHOL) < 0.33, two phases coexist: complex (2:1) plus pure lecithin; when 0.33 < x(CHOL) < 0.67 complexes (2:1) and (1:2) coexist as separate phases. The corresponding phase diagram in water for these mixtures was determined by DSC and isopycnic centrifugation in D(2)O-H(2)O gradients. Aqueous dispersions were prepared by various methods (vortexing, dialysis, sonication) yielding identical results except as noted below. The data presented supports the following phase relations. When x(CHOL) < 0.33, two lipid phases coexist: pure lecithin plus complex (2:1) where the properties of the lecithin phase are determined by whether the temperature is below or above T(c), the gel-liquid crystal transition temperature. Therefore, complex (2:1) will coexist with gel state below T(c) and with liquid crystal above T(c). The densities follow in the order gel > complex (2:1) > liquid crystal. The density of complex (2:1) is less sensitive to temperature in the range 5 degrees -45 degrees C compared to the temperature dependence for DML and DPL where large changes in density occur at T(c). When x(CHOL) > 0.33, CHOL phase coexists with complex (2:1); anhydrous complex (1:2) is apparently not stable in H(2)O. The results are independent of the method and temperature used for preparing the lipid dispersions. However, when dispersions are prepared by sonication or with solvents at T > T(c), an apparent 1:1 complex is formed. Evidence suggests the 1:1 complex is metastable.

Calorimetry, Differential Scanning↗
PubMed1979

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions.

Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.

Acetylcholine↗
PubMed1978

Use of immobilized light-harvesting chlorophyll a/b protein to study the stoichiometry of its self-association.

D. J. Davis & E. L. Gross (1976) Biochim. Biophys. Acta 449, 554-564 previously observed that the light-harvesting chlorophyll a/b protein or chlorophyll protein complex II self-associated as determined by ultracentrifugation. We have determined the stoichiometry of complex formation by immobilizing the monomer on ethylenediamine-Sepharose 4B and determing the ability of immobilized protein to bind the free protein. The amount of soluble protein bound to the immobilized protein increased as the concentration of soluble protein increased. The binding was maximal between pH 7 and 8. The maximum binding was three molecules bound per one molecule of protein immobilized. These results indicate that a tetramer is the intrinsic structural unit of the light-harvesting chlorophyll a/b protein in the chloroplast membrane. Upon complex formation, the chlorophyll fluorescence was decreased without any spectral change. The maximum binding was approximately doubled upon addition of 0.5 mM CaCl2 whereas 5 mM NaCl had no effect. Addition of CaCl2 had no effect on the fluorescence of the monomer. The light-harvesting chlorophyll a/b protein can be isolated from a sodium lauryl sulfate extract of chloroplasts by affinity chromatography using the immobilized light-harvesting chlorophyll a/b protein.

Cations↗
PubMed1977

Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate.

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.

Affinity Labels↗
PubMed1976

Spectroelectrochemical investigations of stoichiometry and oxidation-reduction potentials of cytochrome c oxidase components in the presence of carbon monoxide: the "invisible" copper.

Spectroelectrochemical studies are presented for the carbon monoxide complex of isolated, purified cytochrome c oxidase (EC 1.9.3.1) in solutions saturated with carbon monoxide. The results indicate a stoichiometry of three equivalents per oxidase-carbon monoxide complex molecule. Formal reduction potentials (Eo) of the two copper and one heme component at pH 7.0 were obtained by means of quantitative absorbance-charge titrations in the absence and presence of cytochrome c, and by means of a Nernstian "Minnaert" plot in the presence of cytochrome c. Analysis of the absorbance-charge curves from these titrations gave an indirect determination of the high potential, "invisible" copper component. The copper potentials in the carbon monoxide complex were found to be relatively unchanged with respect to those of the native enzyme. The Eo values obtained were: high potential ("invisible") copper (340 +/- 20 mV (NHE)), low potential copper (190 +/- 20 mV), and low potential heme (250 +/- 10 mV).

Carbon Monoxide↗
PubMed1975

Methylation of the ribosomal proteins in Escherichia coli. Nature and stoichiometry of the methylated amino acids in 50S ribosomal proteins.

Methylated ribosomal proteins from Escherichia coli 50S subunit are localized by growing cells in a medium containing (1-14C)methionine and (3H-methyl)-methionine and comparing the 3H/14C ratio for each of the 50S ribosomal proteins. The following proteins are methylated: L11, L1, L3, L5, L7, L8, L9, L12, L18, and L33. The nature and stoichiometry of the methylated amino acid(s) in each of the methylated proteins are determined. Protein L11 is the most heavily methylated of all the 50S subunit proteins. This protein has previously been implicated in the peptidyl transferase reaction during protein synthesis (K. H. Nierhaus and V. Montejo (1973), Proc. Nat. Acad. Sci. U. S. 47, 1588-1602). Three proteins (L1, L3, and L5) have intermediate levels of methylation and contain about 0.4-0.6 methyl groups each per molecule of protein. Five other proteins (L7, L8, L9, L12, and L18) are also methylated to a slight extent (-0.1 methyl group/molecule of protein). One unknown methylated neutral amino acid was detected in protein L11 and at least one and possibly two other unidentified methylated amino acids appeared to be present in protein L33.

Amino Acids↗
PubMed1975

Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate.

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.

Adenosine Triphosphatases↗
PubMed1977

Stoichiometry of GTP hydrolysis and tubulin polymerization.

Microtubule formation from lamb brain tubulin isolated by affinity chromatography and freed of exchangeable nucleotide requires GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethylenediphosphate and guanylylimidodiphosphate fail to replace GTP; in addition, neither the presence of microtubule associated proteins nor 5 M glycerol relieves the GTP requirement. The relation of GTP concentration and microtubule formation shows an association constant K = 1 X 10(4) M-1; furthermore, GDP and guanylylimidodiphosphate are competitive inhibitors of GTP for polymerization. Using a rapid filter assay for microtubule formation that allows the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of two molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules has been found.

Animals↗
PubMed1978

Kinetics and stoichiometry of Na-dependent Li transport in human red blood cells.

This paper describes the kinetics and stoichiometry of a tightly coupled Na-Li exchange transport system in human red cells. The system is inhibited by phloretin and furosemide but not by ouabain. Li influx by this system increases and saturates with increasing concentrations of external Li and internal Na and is inhibited competitively by external Na. Comparable functions relate Li efflux and Na efflux to internal and external Li and Na concentrations. Analysis of these relations yields the following values for the ion concentrations required to half-maximally activate the transport system: internal Na and Li 9.0 and 0.5 mM, respectively, external Na and Li 25 and 1.5 mM, respectively. The system performs a 1:1 exchange of Na and Li moving in opposite directions across the red cell membrane. We found no evidence for a simultaneous transport of more than one Na and Li by the system. The maximum transport rate of Na-dependent Li transport varied between 0.1 and 0.37 mmol/(liter of cells X h) in the red cells of the five normal male subjects studied. No significant variations between individual subjects were observed for bicarbonate-stimulated Li transport and for the residual Li fluxes which occur in the absence of bicarbonate and in the presence of ouabain plus phloretin.

Biological Transport, Active↗
PubMed1977

The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.

The stoichiometry of inhibition and binding of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor produced by Streptomyces albogriseolus S-3253 (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067) against subtilison BPN' [EC 3.4.21.14] was studied. The inhibition of the hydrolysis of p-nitrophenyl acetate by subtilisin BPN' was measured both at the pre-steady state with a stopped-flow apparatus and at the steady state. The stopped-flow study demonstrated the disappearance of the initial burst of the enzyme reaction. The ultraviolet absorption difference spectra observed on mixing the inhibitor and the enzyme suggested changes in the environment of tryptophyl and tyrosyl residues in the proteins. Titration by means either of the degree of inhibition at the steady state or of the magnitude of the ultraviolet difference absorbance revealed that the inhibitor (dimer, MW: 23,000) bound and inhibited two molecules of subtilisin BPN'. The inhibitor constant, Ki, against subtilisin BPN' was estimated to be less than 10(-9)M at pH 8.50. The type of inhibition of this inhibitor is discussed.

Bacterial Proteins↗
PubMed2026

Estimation of chloroplast macromolecular complex copy numbers and subunit stoichiometries during the Chlamydomonas reinhardtii cell cycle.

An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg&#x2009;cell-1 of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis, and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05&#x2009;&#xd7;&#x2009;106 molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08&#x2009;&#xd7;&#x2009;106 complexes per cell. PSI averages 1.75&#x2009;&#xd7;&#x2009;106 complexes per cell and cytochrome b6f averages 0.77&#x2009;&#xd7;&#x2009;106 complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artifacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Chlamydomonas reinhardtii↗
PubMed1977

Proteins of Rous-associated virus type 61: polypeptide stoichiometry and evidence that glycoprotein gp35 is not a cleavage product of gp85.

The two glycoproteins, gp85 and gp35, of Rous-associated virus type 61 (RAV-61), were isolated from radiolabeled virions by gel electrophoresis and digested with trypsin. The chromatographic profile of the gp35 digest revealed no peaks in common with that of gp85; therefore, the smaller glycoprotein is not a cleavage product of gp85. The stoichiometry of radiolabeled RAV-61 proteins was studied by quantitative gel filtration and gel electrophoresis. Among the 11 polypeptides identified were 4 minor ones, including the beta(p91) and alpha(p64) chains of reverse transcriptase and two unidentified chains, p76 and p35; the latter two were unmasked by removing the virions' surface glycoproteins with a protease, bromelain. Virions contained some 15 to 30 molecules of reverse transcriptase.

Animals↗
PubMed1978

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.

Binding Sites↗
PubMed2026

SpxA1 and SpxA2 function as a stoichiometry-dependent regulatory rheostat governing virulence gene expression in group A Streptococcus.

UNLABELLED: Group A Streptococcus (GAS) is a human-restricted pathogen whose global incidence has surged in the post-COVID era. The ability of GAS to shift from a colonizing to invasive phenotype depends on coordinated virulence gene regulation in response to host-derived signals. However, the mechanisms by which individual stress-sensing systems interact to reshape the virulence gene regulatory landscape remain incompletely understood. Here, we define the regulatory programs of two conserved transcriptional regulator paralogs, SpxA1 and SpxA2, using an integrated multi-omic approach combining RNA-seq, data-independent acquisition proteomics, NanoString-based transcriptional profiling across multiple host-relevant stress conditions, and chromatin immunoprecipitation with exonuclease treatment (ChIP-exo). RNA-seq revealed functionally distinct regulons with SpxA1 governing oxidative stress defense and SpxA2 coordinating virulence-associated gene expression linked to the CovRS two-component regulatory system. Proteomic analysis established SpxA2 as a ClpXP protease substrate in GAS and identified reciprocal paralog accumulation upon loss of either SpxA1 or SpxA2, consistent with compensatory transcriptional upregulation. NanoString profiling under bacitracin and human neutrophil peptide-1 challenge identified four gene modules with distinct stoichiometry-dependent and condition-dependent regulatory logic, revealing that the SpxA1/SpxA2 ratio rather than the activity of either paralog alone determines which transcriptional programs are engaged. ChIP-exo demonstrated that SpxA2 directly modulates CovR-DNA binding occupancy in a CovR-binding motif-dependent manner, simultaneously antagonizing CovR dimer binding at an extended (25 bp) CovR motif and facilitating CovR monomer binding at the canonical ATTARA motif. These findings establish the LiaFSR-SpxA2-CovRS axis as a cross-regulatory circuit through which GAS cell envelope stress sensing is directly transduced into coordinated virulence gene regulatory changes. IMPORTANCE: Group A Streptococcus (GAS) causes millions of infections annually, including a recent global surge in invasive disease. To survive in the human host, GAS must rapidly reprogram virulence gene expression in response to host-derived stresses. This study characterizes two conserved transcriptional regulators, SpxA1 and SpxA2, that govern this response through interaction with RNA polymerase to indirectly influence the DNA-binding activity of downstream transcription factors. We show that SpxA2, activated by a cell envelope stress-sensing system responding to human antimicrobial peptides, reshapes the binding of the master virulence regulator CovR in a promoter-specific manner, coupling cell envelope stress sensing to virulence gene regulation. The stoichiometric balance between SpxA1 and SpxA2 functions as a regulatory rheostat calibrating overall virulence gene regulatory tone, providing a framework for understanding how RNA polymerase-interacting regulators coordinate stress responses and virulence gene control across Gram-positive bacterial pathogens.

Streptococcus pyogenes↗
PubMed1975

The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. II. Investigations on films containing condensed or swollen chicken erythrocyte nuclei.

As models for different states of chromatin compactness, nuclei from chicken erythrocytes were isolated and either osmotically swollen or kept as condensed as possible. Both types of nuclei were then fixed and incorporated into polyacrylamide films. Hydrolysis with 5 N HCl and staining with Schiff's reagent of these model films were studied using several parameters. The phosphate content of the films was analyzed as a parameter for the depolymerization losses and the staining with Schiff's reagent as a parameter for the apurinic acid (APA) content. The loss of ultraviolet absorbance from the films and the accumulation of ultraviolet absorbing substances in the hydrolyzing acid were monitored as parameters for the progress of hydrolysis. Conversion of the generated aldehyde groups to APA-Schiff chromophore is shown to take place with the same stoichiometry for both types of nuclei as well as for DNA in model films. It is further shown that the nuclei- and DNA-films are suitable models for investigating the influence of chromatin compactness on the course of the Feulgen-Schiff reaction. For the most compact form of chromatin studied, a very high reduction in staining intensity of up to 40% could be demonstrated after certain normally applied hydrolysis times. This is due primarily to a decrease with a factor of 2.3 of the depurination rate constants of these models (from 0.030/min to 0.013/min). Therefore prolonged hydrolysis periods are required to obtain the same APA concentrations, but then depolymerization processes cause losses of nuclear material. The differences in depurination rates could be explained by a decrease in [H3O]+ in the neighborhood of the purine-sugar linkages, caused by the presence of fixed positive charges form the protein components of the chromatin. These findings may explain the cytophotometrically determined differences in chromophore yield of 10-20% found in the nuclei of cells with different states of compactness of their chromatin. The descending part of the Feulgen hydrolysis curve represents the depolymerization of APA and loss by diffusion of the reaction products. In the Appendix, cytophotometric data of cells have been analyzed to show that this part of the hydrolysis curve may be used to estimate the acid stability of chromatin complexes. The depurination and depolymerization rates found closely correspond with the data obtained from the model films.

Animals↗
PubMed1977

The TcO4-binding to human serum albumin --the stoichiometry and characteristics of the binding equilibrium.

The stoichiometry and the characteristics of the TcO4-binding equilibrium to human serum albumin were investigated by the use of 99TcO4-. Based on the Scatchard plots, the number of binding sites and the association constants were obtained at pH 7.38,6.04, 5.10, 4.18, 2.80 and 0.65, respectively. From the parameters at pH 7.38, it was estimated that 64% of TcO4- added was bounded to human serum albumin under physiological condition. Variance in the values at pH 7.38, 6.04 and 5.10 shows that these bindings are stabilized by electrostatic forces. Below pH 4.18, the number of binding sites increased and the association constants constants diminished. These phenomena may be attributed to the conformation change of human serum albumin.

Binding Sites↗
PubMed1975

Stoichiometry of compounds bound to human erythrocytes in relation to morphology.

Most work on human erythrocyte interaction with drugs and other compounds has been reported on the basis of total concentrations. Total concentrations alone do not reveal numbers of molecules bound per cell, v. This paper emphasizes determination of v and of binding isotherms, in conjunction with changes in cell morphologies and in hypotonic shock behavior as v is varied. Four drugs and five other compounds were studied, with fresh erythrocytes. The principal findings are: (1) the intact erythrocyte engages in two kinds of binding mechanisms, statistical binding and cooperative binding, depending on the compound. In the case of a detergent, dodecylbenzene sulfonate, the binding is nearly quantitative. (2) The compounds often induce considerable protection against hypotonic hemolysis. However, the binding levels at which maximum protection occurs are rather close to the levels, vL, that occur upon complete conversion to the first distorted morphology. Therefore, the maximally protected erythrocyte may be a distorted erythrocyte. (3) The value n is the apparent total number of sites from Scatchard plotting for compounds which bind in a statistical manner. Levels vp and vw characterize maxima in cooperative binding behavior, also from Scatchard plotting of the data. Despite the wide diversity of over-all levels at which compounds exert their effects, the critical binding levels of and numbers of sites fall into a narrow range:n, vL, vP, and vw are all between 1 and 8 times 10-7 molecules or sites per cell. Most of our data, and that from some other laboratories, indicate that about 2 plus and minus 1 times 10-7 sites per erythrocyte are available for compound binding by the intact cell. Beyond that level, the cell in suspension almost always will be forced into the first obvious morphology change, as seen by phase contrast microscopy. (4) Once stoichiometries are established, the total binding capacity of erythrocytes for such compounds, in blood, can be estimated. An intruding organic molecule would encounter about 6 times as many plasma albumin sites as erythrocyte sites, if the plasma albumin sites were free. However, because albumin in vivo usually forms a complex with one to two fatty acids, the erythrocyte itself is rather likely to act as a transport particle for such compounds.

Binding Sites↗