Paramyxovirus membrane protein enhances antibody production to new antigenic determinants in the actin molecule: a model for virus-induced autoimmunity.

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.     

Apolar interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles

The interaction of bovine pancreatic alpha-chymotrypsin with dimyristoyl phosphatidylcholine (PC) vesicles was measured turbimetrically. The protein interacted with the vesicles at NaCl concentrations of above 0.8 M. The turbidity reached a plateau on increase in the amount of either the protein or the vesicles in the presence of a fixed amount of the other component. The precipitates formed contained both PC and protein in ratios varying with the initial amount of each component. On mixing chymotrypsin and PC vesicles, time-dependent turbidity increase was high at below pH 2.5, but relatively small at neutral and alkaline pH values. Apolar interaction between the two components was confirmed by demonstrating an increase in fluorescence intensity of chymotrypsin in the presence of the PC in 1 M NaCl. The turbidity of a mixture of PC vesicles and bovine serum albumin (BSA) increased even in the absence of 1 M NaCl, whereas the turbidities of mixtures of the vesicles and lysozyme or alpha-lactalbumin did not change with time in the presence of 1 M NaCl at pH 8.0.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Interactions between globular proteins and F-actin in isotonic saline solution.

Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.     

Protein-phosphate complexes as an inhibitor of iron-induced lipid peroxidation

Effect of phosphate buffer (pH 6.2) alone or in the presence of bovine serum albumin and other proteins on iron (II)-induced lipid peroxidation was studied. Phosphate buffer alone and in the presence of bovine serum albumin was found to inhibit lipid peroxidation. The inhibition was higher when bovine serum albumin was also present. Other proteins also inhibited lipid peroxidation in the presence of phosphate. Inhibition by proteins in the presence of phosphate seems to be due to binding of iron with phosphate and with protein-phosphate complexes. Reversal in inhibition was observed with an increase in iron concentration in reaction mixture. Equilibrium dialysis showed more binding of iron to protein in the presence of phosphate than in the presence of chloride ions.     

Circular dichroic study of the conformational stability of sulfhydryl-blocked bovine serum albumin

1. The blockage of the single sulfhydryl-group of bovine serum albumin does not alter the secondary structure, although the alpha-helical structure is destabilized since lower concentrations of guanidine and of urea unfold the protein. 2. What happens to the previously helical structure depends upon the reagent used to block the sulfhydryl-group. Bovine serum albumin derivatized with 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate preferentially acquire the beta-structure in high concentrations of guanidine and urea, whereas iodoacetamide-derivatized bovine serum albumin acquires primarily the random coil structure. 3. Part of the helical structure is also lost in 5-6 mM sodium dodecyl sulfate; thionitrobenzoate-bovine serum albumin shows an increase in the random coil, whereas the two alkylated proteins display the increase both in beta-structure and random coil. 4. Carboxymethylation or carboxamidomethylation of fully reduced bovine serum albumin results in a drastic change in the secondary structure of the protein with a substantial decrease in alpha-helix and a corresponding increase in both beta-structure and random coil. These extensively alkylated proteins also display differences in denaturation profiles in solutions of guanidine and urea.     

Effect of Fetal Bovine Serum Glycoproteins on the In Vitro Proliferation of the Oyster Parasite Perkinsus marinus: Development of a Fully Defined Medium

ABSTRACT. The oyster parasite Perkinsus marinus replicates in our medium consisting of Dulbecco modified Eagle's medium: Ham's F12 nutrient mixture (1:1) supplemented with 1–5% fetal bovine serum, with a doubling time of 24 hours during the exponential phase of the culture. Fetal bovine serum concentrations above 5% dramatically reduced parasite proliferation in a dose-dependent manner. We tested the individual effects of the three major protein components of fetal bovine serum (fetuin, transferrin and albumin) on the replication of the parasite in a serum-free medium. At the concentrations tested, fetuin enhanced parasite growth, whereas albumin had a modest positive effect and transferrin was inhibitory. Proteolytic digestion of fetuin, strongly diminished its growth-enhancing properties, indicating that the overall glycoprotein architecture may be required for activity. On the contrary, desialylation of fetuin slightly enhanced its growth-promoting activity. The addition of fetuin at 1.7 mg/ml to the serum-free DME: Ham's F12 medium yielded growth rates that are comparable to those obtained with our standard culture methodology. This has resulted in a fully defined culture medium that will allow for a rigorous characterization of excretory/secretory products involved in modulating or blocking the host's humoral and cellular defense mechanisms.     

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Partition of whey milk proteins in aqueous two-phase systems of polyethylene glycol-phosphate as a starting point to isolate proteins expressed in transgenic milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha lactoalbumin and beta lactoglobulin) and alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000; 1500 and 3350)-potassium phosphate was analysed. Bovine serum albumin and alpha lactoalbumin concentrated in the polyethyleneglycol rich phase with a partition coefficient of 10.0 and 27.0, respectively, while beta lactoglubulin and alpha-1 antitrypsin showed affinity for the phosphate-rich phase with a partition coefficient of 0.07 and 0.01, respectively. An increase of medium pH induced an increase of the partition coefficient of these proteins while the increase in polyethyleneglycol molecular mass induced the opposite behaviour. The system polyethyleneglycol 1500-pH 6.3 showed the best capacity for recovering the alpha-1 antitrypsin with a yield of 80% and a purification factor between 1.5 and 1.8 from an artificial mixture of the milk whey proteins and alpha-1 antitrypsin. The method appears to be suitable as a starting point to isolate proteins expressed in transgenic milk.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

Formation of Molecular Complexes Between a Structurally Defined M Protein and Acylated or Deacylated Lipoteichoic Acid of Streptococcus pyogenes

The orientation of lipoteichoic acid (LTA) molecules on the surface of bacterial cells undoubtedly is determined by the ability of the LTA, during its transit through the cell wall, to bind via its polyglycerophosphate backbone or its glycolipid moieties to other constituents of the cytoplasmic membrane and the cell wall. We have investigated the possibility that LTA may become anchored to the cell surface by binding through its polyanionic backbone to positively charged regions of cell wall proteins. LTA was found to prevent the precipitation of partially purified HCl extracts of several strains of streptococci as well as a structurally defined streptococcal M protein molecule (pep M24) in 83% solutions of ethanol. The formation of complexes between LTA and M protein was demonstrated further by immunoelectrophoresis of pep M24 protein with increasing concentrations of radiolabeled LTA and by using antiserum against pep M24 to develop precipitin arcs. Pep M24 electrophoresed alone produced a single precipitin arc close to the origin. In contrast, when electrophoresed as a mixture with LTA or deacylated LTA, the M protein produced a second precipitin arc toward the anode coinciding with the area of migration of the radioactive LTA. Increasing concentrations of LTA or deacylated LTA shifted increasing amounts of the pep M24 antigen to the region of the second arc. Maleylation of M protein to block the positively charged free amino groups before mixing it with LTA prevented the formation of complexes. The complexes formed by the M protein with LTA, but not with deacylated LTA, showed the capacity to bind bovine serum albumin; LTA had been shown previously to bind to the fatty acid binding sites on bovine serum albumin. These results indicate that the LTA molecule is able to bind via its polyanionic backbone to positively charged residues of surface proteins of cells of S. pyogenes. The implications of such interaction as to the orientation of LTA molecules on the surface of cells of S. pyogenes are discussed.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Protein tyrosine phosphatase activity regulates endothelial cell-cell interactions,the paracellular pathway,and capillary tube stability

Protein tyrosine phosphorylation is tightly regulated through the actions of both protein tyrosine kinases and protein tyrosine phosphatases. In this study, we demonstrate that protein tyrosine phosphatase inhibition promotes tyrosine phosphorylation of endothelial cell-cell adherens junction proteins, opens an endothelial paracellular pathway, and increases both transendothelial albumin flux and neutrophil migration. Tyrosine phosphatase inhibition with sodium orthovanadate or phenylarsine oxide induced dose- and time-dependent increases in [14C]bovine serum albumin flux across postconfluent bovine pulmonary artery endothelial cell monolayers. These increases in albumin flux were coincident with actin reorganization and intercellular gap formation in both postconfluent monolayers and preformed endothelial cell capillary tubes. Vanadate (25 microM) increased tyrosine phosphorylation of endothelial cell proteins 12-fold within 1 h. Tyrosine phosphorylated proteins were immunolocalized to the intercellular boundaries, and several were identified as the endothelial cell-cell adherens junction proteins, vascular-endothelial cadherin, and beta-, gamma-, and p120-catenin as well as platelet endothelial cell adhesion molecule-1. Of note, these tyrosine phosphorylation events were not associated with disassembly of the adherens junction complex or its uncoupling from the actin cytoskeleton. The dose and time requirements for vanadate-induced increases in phosphorylation were comparable with those defined for increments in transendothelial [14C]albumin flux and neutrophil migration, and pretreatment with the tyrosine kinase inhibitor herbimycin A protected against these effects. These data suggest that protein tyrosine phosphatases and their substrates, which localize to the endothelial cell-cell boundaries, regulate adherens junctional integrity, the movement of macromolecules and cells through the endothelial paracellular pathway, and capillary tube stability.     

Actin is a noncompetitive plasmin inhibitor.

Actin, one of the most abundant cellular proteins, circulates at micromolar concentrations in peripheral blood. Because actin released from dying cells may be trapped in fibrin clots that form at sites of tissue injury, we examined the effects of actin upon lysis of fibrin clots in vitro. Incorporation of native rabbit skeletal muscle actin into fibrin clots slowed their rates of lysis for periods of up to 24 h, an effect not seen when comparable concentrations of human IgG or bovine serum albumin were added instead. Actins isolated from a variety of sources inhibited plasmin's hydrolysis of the synthetic substrate S-2251 in a noncompetitive manner, with a Ki of a 0.6-3.1 microM. Inhibition was rapid, but covalent actin-plasmin complexes were not formed. Both epsilon-aminocaproic acid and tranexamic acid prevented actin's inhibition of plasmin, suggesting that accessible lysine residues of actin interact with the kringle (lysine-binding) regions of plasmin. Neither of the high-affinity actin-binding proteins of plasma (plasma gelsolin and vitamin D-binding protein) prevented actin from inhibiting plasmin. These findings suggest that actin released into the extracellular space following cell death may modulate plasmin action, and hence a number of plasmin-dependent biological responses, at sites of inflammation and tissue injury.     

Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin,alpha-globulins,alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6

The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.     

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.     

Protein adsorption at solid-liquid interfaces: Part II--Adsorption from binary protein mixture

Extent of adsorption of proteins at alumina-water interface from solutions containing binary mixture of beta-lactoglobulin and bovine serum albumin (BSA), beta-lactoglobulin and gelatin, and gelatin and bovine serum albumin has been estimated as functions of protein concentrations at varying pH, ionic strength, temperature and weight fraction ratios of protein mixture. The extent of adsorption (gamma lacw) of lactoglobulin in the presence of BSA increases with increase of protein concentration (Clac) until it reaches a maximum but a fixed value gamma lacw(m). Extent of adsorption gamma serw also initially increases with increase of protein concentrations until it reaches maximum value gamma serw(m). Beyond these protein concentrations, adsorbed BSA is gradually desorbed due to the preferential adsorption of lactoglobulin from the protein mixture. In many systems, gamma serw at high protein concentrations even becomes negative due to the strong competition of BSA and water for binding to the surface sites in the presence of lactoglobulin. For lactoglobulin-gelatin mixtures, adsorption of both proteins is enhanced as protein concentration is increased until limiting values for adsorption are reached. Beyond the limiting value, lactoglobulin is further accumulated at the interface without limit when protein concentration is high. For gelatin-albumin mixtures, extent of gelatin adsorption increases with increase in the adsorption of BSA. The limit for saturation of adsorption for gelatin is not reached for many systems. At acid pH, adsorbed BSA appears to be desorbed from the surface in the presence of gelatin. From the results thus obtained the role of electrostatic and hydrophobic effects in controlling the adsorption process has been analysed.     

Interaction of a protein, BSA, and a fluorescent probe, Mag-Indo-1, influence of EDTA and calcium on the equilibrium.

Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore.