Adsorption of globular proteins on locally planar surfaces: models for the effect of excluded surface area and aggregation of adsorbed protein on adsorption equilibria.

Equilibrium statistical-thermodynamic models are presented for the surface adsorption of proteins modeled as regular convex hard particles. The adsorbed phase is treated as a two-dimensional fluid, and the chemical potential of adsorbed protein is obtained from scaled particle theory. Adsorption isotherms are calculated for nonassociating and self-associating adsorbing proteins. Area exclusion broadens adsorption isotherms relative to the Langmuir isotherm (negative cooperativity), whereas self-association steepens them (positive cooperativity). The calculated isotherm for adsorption of hard spheres using scaled particle theory for hard discs agrees well with that calculated from the hard disc virial expansion. As the cross section of the adsorbing protein in the plane of the surface becomes less discoidal, the apparent negative cooperativity manifested in the isotherm becomes more pronounced. The model is extended to the case of simultaneous adsorption of a tracer protein at low saturation and a competitor protein with a different size and/or shape at arbitrary fractional saturation. Area exclusion by competitor for tracer (and vice versa) is shown to substantially enhance the displacement of tracer by competitor and to qualitatively invalidate the standard interpretation of ligand competition experiments, according to which the fractional displacement of tracer by competitor is equal to the fractional saturation by competitor.     

Effects of excluded surface area and adsorbate clustering on surface adsorption of proteins I. Equilibrium models

Statistical-thermodynamic models for the equilibrium adsorption of proteins onto homogeneous, locally planar surfaces are presented. An extension of earlier work [R.C. Chatelier, A.P. Minton, Biophys. J. 71 (1996) 2367], the models presented here allow for the formation of a broadly heterogeneous population of adsorbate clusters in addition to excluded volume interactions between all adsorbate species. Calculations are carried out for three simple models for the structure of adsorbate, illustrating similarities and differences in the equilibrium properties of maximally compact clusters, minimally compact clusters and isomerizing clusters. Depending upon the strength of attractive interactions between adsorbate molecules, the resulting equilibrium isotherms may exhibit negative cooperativity, positive cooperativity, essentially no apparent cooperativity, or a mixture of positive cooperativity at low surface density and negative cooperativity at high surface density of adsorbate. The condition of apparent lack of cooperativity, which might naively be interpreted as evidence of a lack of interaction between adsorbate molecules, actually conceals a balance between attractive and repulsive interactions and extensive clustering of adsorbate.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Nonfouling behavior of polycarboxybetaine-grafted surfaces: structural and environmental effects

Zwitterionic carboxybetaine (CB) has unique dual functionality for ligand immobilization on a nonfouling background. The properties of CB groups depend on their spacer groups between the positive quaternary amine groups and the negative carboxyl groups and environmental factors (e.g., ionic strengths and pH values). In this work, five polycarboxybetaines were prepared, including one polycarboxybetaine methacrylate (polyCBMA) and four polycarboxybetaine acrylamides (polyCBAAs) with different spacer groups. The polymers were grafted from a gold surface covered with initiators using surface-initiated atom transfer radical polymerization. Fibrinogen adsorption was measured as a function of ionic strengths and pH values using surface plasmon resonance sensors. The responsive protein adsorption on four polyCBAAs was mapped out. Results show that most of these surfaces exhibit high protein resistance in a wide range of ionic strengths and are more effective than zwitterionic self-assembled monolayers. Although protein adsorption tends to increase at low ionic strength and low pH value, it is still very low for polycarboxybetaines with a methylene, an ethylene, or a propylene spacer group but is more evident for polyCBAA with a longer spacer group (i.e., a pentene group). The response to ionic strengths and pH values can be attributed to the antipolyelectrolyte and protonation/deprotonation properties of polycarboxybetaines, respectively. Both of these properties are related to the spacer groups of CBs.     

Protein Adsorption on Erythrocytic Membranes and Its Effect on Erythrocyte Rheology in Athletes during Competition Exercise

The adsorption of high-molecular-weight plasma proteins on erythrocyte membranes was studied in athletes after prolonged exercise under competition conditions. The adsorption of individual high-molecular-weight protein fractions depended on their concentration. The adsorption index changed biphasically at submaximum exercise. The adsorption of plasma high-molecular-weight protein fractions was associated with the fluidity of concentrated erythrocyte suspensions. The adsorbed high-molecular-weight globulins and fibrinogen had different effects on the parameters of erythrocyte rheology.     

Effects of excluded surface area and adsorbate clustering on surface adsorption of proteins. II. Kinetic models

Models for equilibrium surface adsorption of proteins have been recently proposed (Minton, A. P., 2000. Biophys. Chem. 86:239-247) in which negative cooperativity due to area exclusion by adsorbate molecules is compensated to a variable extent by the formation of a heterogeneous population of monolayer surface clusters of adsorbed protein molecules. In the present work this concept is extended to treat the kinetics of protein adsorption. It is postulated that clusters may grow via two distinct kinetic pathways. The first pathway is the diffusion of adsorbed monomer to the edge of a preexisting cluster and subsequent accretion. The second pathway consists of direct deposition of a monomer in solution onto the upper (solution-facing) surface of a preexisting cluster ("piggyback" deposition) and subsequent incorporation into the cluster. Results of calculations of the time course of adsorption, carried out for two different limiting models of cluster structure and energetics, show that in the absence of piggyback deposition, enhancement of the tendency of adsorbate to cluster can reduce, but not eliminate, the negative kinetic cooperativity due to surface area exclusion by adsorbate. Apparently noncooperative (Langmuir-like) and positively cooperative adsorption progress curves, qualitatively similar to those reported in several published experimental studies, require a significant fraction of total adsorption flux through the piggyback deposition pathway. According to the model developed here and in the above-mentioned reference, the formation of surface clusters should be a common concomitant of non-site-specific surface adsorption of proteins, and may provide an important mechanism for assembly of organized "protein machines" in vivo.     

Membrane cooperative enzymes. High molecular specificity for blocking action of thyroxine on triiodothyronine effect in rat erythrocyte and Escherichia coli systems.

The molecular specificity for the blocking action of thyroxine on the triiodothyronine effect in the cooperativity of membrane-bound rat erythrocyte acetylcholinesterase and Escherichia coli Ca2+-ATPase was analyzed. Changes in the values of n (Hill coefficient) were obtained at strict physiological levels of these hormones. The structural requirements of the thyroid hormones to modify the membrane-bound systems were studied using various analogues of these hormones. In the erythrocyte system, a very high molecular specificity for triiodothyronine and thyroxine actions was found. The L-alanine side is essential to carry out both the allosteric desensitization and the blocking effects. The blocking ability of thyroxine is characterized by the presence of iodine in the 5' position. The bacterial system presented only specificity for the triiodothyronine allosteric desensitization. A system of membrane-bound enzymes for the study of the actions of thyroid hormones, is presented here.     

Effect of Adsorbed Proteins on the Rheological Properties of Erythrocytes

Impedance spectroscopy in the radio-frequency band was used to probe the adsorptive properties of red blood cells. Fluidity assays of concentrated erythrocyte suspensions revealed a close relationship between the protein adsorption on erythrocyte membranes and the extent of cell interaction in the flow. Patients with impaired peripheral circulation displayed an increase in adsorption of high-molecular-weight proteins and a dramatic decrease in fluidity of erythrocyte suspensions. Hence, adsorption was assumed to contribute to rheological disorders.     

Ghost cells as sorption matrix for virus concentration from water

Pig erythrocyte membranes were used as adsorbent material for the concentration of bacteriophage phi x-174, MS-2, and f2 from 5 ml of saline solution. The adsorption was carried out at pH 3.5, and the elution was carried out at pH 7.0. Compared with adsorption on 3% beef extract, bacteriophage adsorption on erythrocyte ghost cells yielded 93 to 100%, and elution was 92 to 100%, whereas beef extract organic flocculation yielded adsorption of 0 to 98.8% and elution of 61 to 86.6%. The same procedure but with poliovirus LSC-1 gave 100% adsorption and 91 to 129% elution.     

Ghost cells as sorption matrix for virus concentration from water.

Pig erythrocyte membranes were used as adsorbent material for the concentration of bacteriophage phi x-174, MS-2, and f2 from 5 ml of saline solution. The adsorption was carried out at pH 3.5, and the elution was carried out at pH 7.0. Compared with adsorption on 3% beef extract, bacteriophage adsorption on erythrocyte ghost cells yielded 93 to 100%, and elution was 92 to 100%, whereas beef extract organic flocculation yielded adsorption of 0 to 98.8% and elution of 61 to 86.6%. The same procedure but with poliovirus LSC-1 gave 100% adsorption and 91 to 129% elution.     

Activation of the purified erythrocyte plasma membrane Ca2+- ATPase by organic solvents

In this report it is shown that organic solvents mimic the stimulatory effects of calmodulin and acidic phospholipids on the erythrocyte plasma membrane Ca2+-ATPase. The solvents used were dimethyl sulfoxide (20%, v/v), glycerol (20% v/v), ethylene glycol (20%, v/v) and polyethylene glycol (Mr 6000-8000) (10%, w/v). These solvents increased both the affinity for Ca2+ and the turnover number of the enzyme. The increase in Ca2+ affinity is additive to that achieved with calmodulin. The calcium cooperativity observed in the presence of calmodulin disappears after the addition of dimethyl sulfoxide to the medium. The present data support the proposal that activation of the erythrocyte plasma membrane Ca2+-ATPase is promoted by hydrophobic interactions along the enzyme molecule.     

The Interaction of the Polyene Antibiotic Lucensomycin with Cholesterol in Erythrocyte Membranes and in Model Systems: II. Cooperative Effects in Erythrocyte Membranes

Fluorometric titration curves of erythrocyte membranes with increasing lucensomycin have a sigmoid shape. This behavior, which was not present when colloidal cholesterol suspensions were used, is, however, not peculiar to the membrane structure, being also present in cholesterol-containing phospholipid micelles. Addition of acetic acid induced or increased sigmoidicity. This behavior can either be due to a true cooperativity in binding or to different fluorescence yields of the various lucensomycin-membrane complexes. The latter hypothesis appears to be slightly favored.     

Thermal stabilities of brain spectrin and the constituent repeats of subunits

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (alphaII and betaII) with those of erythrocyte spectrin (alphaI and betaI). The unfolding transition midpoints (T(m)) of the 36 alphaII- and betaII-spectrin repeats extend between 24 and 82 degrees C, with an average higher by some 10 degrees C than that of the alphaI- and betaI-spectrin repeats. This difference is reflected in the T(m) values of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin exhibited strong cooperative coupling, with elevation of the T(m) of the less stable partner corresponding to coupling free energies of approximately -4.4 and -3.5 kcal/mol. The third tandem-repeat construct, by contrast, exhibited negligible cooperativity. Tandem-repeat mutants, in which a part of the "linker" helix that connects the two domains was replaced with a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus, the linker regions, which tolerate few point mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in alphaII- and betaII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin.     

Architecture of the Streptomyces lividans DnaA protein-replication origin complexes

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

The plasticity of estrogen receptor-DNA complexes: binding affinity and specificity of estrogen receptors to estrogen response element half-sites separated by variant spacers

The consensus estrogen response element (cERE) contains a palindromic sequence of two 6-base pair (bp) half-sites separated by a spacer size of 3bp. This study investigates the extent to which estrogen receptors, ERalpha and ERbeta can bind target sequences not considered as conventional EREs. We determined the effect of spacer size (n=0-4) on the binding affinity and conformation of ERalpha and ERbeta in these complexes and the effect of HMGB1 on the complexation. We find (1) both receptors bind similarly and with progressively reduced affinity to cEREn, as n differs from 3; (2) however, both receptors bind as strongly to the cERE with no spacer (cERE0) as to cERE3; (3) HMGB1 enhances ER binding affinity in all complexes, resulting in strong and comparable binding affinities in all complexes examined; (4) the full-length ER binding differs strikingly from similar binding studies for the ER DNA binding domain (ERDBD), with the full-length ER dimer exhibiting strong binding affinity, enormous plasticity and retaining binding cooperativity as the spacer size varies; (5) both protease digestion profiles and monoclonal antibody binding assays indicate the conformation of the receptor in the ER/ERE complex is sensitive to the spacer size; (6) the ER/cERE0 complex appears to be singularly different than the other ER/cEREn complexes in binding and conformation. This multifaceted approach reinforces the notion of the plasticity in ER binding and leads to the hypothesis that in most cases, the minimum requirement for estrogen receptor binding is the ERE half-site, in which one or more cofactors, such as HMGB1, can cooperate to decrease ER binding specificity, while increasing its binding affinity.     

Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family.

The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.