Protein Concentration Dependence Research Articles

Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies

The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 Å, the casein micelles. Removal of Ca 2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H 2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D 2O was used instead of H 2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, ν , and correlation times, τ c , were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and ν was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though τ c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.

The kinetics of bovine growth hormone folding are consistent with a framework model.

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.

Crystal growth studies of lysozyme as a model for protein crystallization

The protein concentration dependence of the growth rate of lysozyme crystals (tetragonal form) was investigated at two NaCl concentrations (3.5%, 5% w/v). The rates for different crystallographic faces were found to have different concentration dependences, resulting in a changing crystal shape with concentration. The experimental results were compared with standard crystal growth theories. The strong concentration dependence could be fitted at high supersaturation by a two-dimensional nucleation model, whereas at low supersaturation a correlation of growth rate with etch-revealed imperfections on the crystals suggested a defeat-mediated growth mechanism. Crystals grown for long times in unstirred solutions exhibited a non-uniform, “patchy” growth when the supersaturation was raised, indicating an inactivation of parts of the crystal surface. This phenomenon may be related to the “cessation of growth”.

Intramolecular electron and proton transfer in proteins: CO2- reduction of riboflavin binding protein and ribonuclease A.

The formate radical (CO2-) reacts with ribonuclease A to form the cystine disulfide radical as one of the products. CO2- reacts with the riboflavin binding protein of chicken egg white with the ultimate product being the neutral flavin semiquinone. Formation of the disulfide radical in ribonuclease is slower than the reaction between protein and CO2-; formation of the flavin semiquinone in the riboflavin binding protein is slower than the protein-CO2- reaction. We conclude for both proteins that CO2- must reduce an as yet unidentified group or groups, which in turn reduce(s) the disulfide of RNase or the flavin of riboflavin binding protein. This conclusion is supported in the case of ribonuclease by the observation of a transient, broad absorption band centered between 350 and 370 nm. The CO2--initiated reductions of the disulfide in ribonuclease and the flavin in the riboflavin binding protein are mixed first- and second-order processes. We propose that the transfer of an electron from the unknown intermediate(s) to the final product involves both inter- and intramolecular paths between groups that may not be in van der Waals contact. With the hydrated electron, in contrast to CO2-, as reductant of the riboflavin binding protein, the anionic semiquinone is observed as an intermediate. The anionic semiquinone is then rapidly protonated, yielding the stable neutral semiquinone. From the reaction kinetics and protein concentration dependence, we conclude that a group or groups on the protein donate(s) a proton to the anionic semiquinone by both inter- and intramolecular paths.

Subunit phosphorylation and activation of skeletal muscle phosphorylase kinase by the cAMP-dependent protein kinase. Divalent metal ion, ATP, and protein concentration dependence.

This report provides a characterization of the effects of varying the concentrations of Mg2+, ATP, phosphorylase kinase, and the cAMP-dependent protein kinase on the activation and phosphorylation of phosphorylase kinase. The results show the following. (a) The Km for MgATP2- for the cAMP-dependent protein kinase-catalyzed phosphorylation is decreased by increasing Mg2+, probably as a consequence of decreasing the free ATP:MgATP2- ratio and increasing free Mg2+. (b) Whereas beta subunit phosphorylation of phosphorylase kinase plays a prominent role in determining its activity, alpha subunit phosphorylation can also modulate activity. (c) The phosphorylation of the alpha subunit, which occurs following the initial cAMP-dependent phosphorylation of the beta subunit, is catalyzed by the cAMP-dependent protein kinase and is not a consequence of EGTA-insensitive (or EGTA-sensitive) autophosphorylation occurring as a result of the enhanced phosphorylase kinase activity. (d) The relationship between subunit phosphorylation and phosphorylase kinase activation is complex and particularly dependent upon concentrations of cAMP-dependent protein kinase and phosphorylase kinase in the activation reaction. The data suggest the possibilities that the pathway of phospho-intermediates involved in the activation process probably varies with the activation conditions, that the efficacy of a specific site to be covalently modified is dependent upon the phosphorylation status of other sites, and that the effect of phosphorylation in regulating activity may also be dependent on the phosphorylation status of other sites. It is clear from the data that the activation process for phosphorylase kinase can be very complex, and it is possible that this complexity might have significant physiological ramifications.

Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity.

These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.

Interaction between Hydrophilic Proteins and Nonionic Detergents Studied by Surface Tension Measurements

Abstract The surface tensions of the systems of hexa(oxyethylene) dodecyl ether (C12E6) vs. lysozyme (Lyz) or bovine serum albumin (BSA) were measured for constructing the binding isotherms, since the surface tension method is more convenient for the systems of nonionic detergents and proteins than is equilibrium dialysis. The obtained binding isotherms depend on the protein concentration; this effect is much more remarkable for Lyz–C12E6 systems than for BSA–C12E6 ones. The binding isotherms of BSA–C12E6 systems obey the Scatchard equation, and the maximum number and estimated free energy for C12E6 binding to BSA are comparable to those for the system of Triton X-100 and BSA determined by equilibrium dialysis. The agreement suggests that the surface tension method is sound and has advantages over equilibrium dialysis for the estimation of the number of detergent molecules bound to protein. In the systems of Lyz–C12E6, the binding isotherms follow the Freundlich-type equation rather than the Scatchard equation. The remarkable protein concentration dependence of the binding isotherms and this result suggest the occurrence of protein aggregation.

Calcium ion-protein interactions in prothrombin activation.

1. The protein concentration dependence observed in the calcium binding to fragment 1 indicates that calcium-mediated dimerization is responsible for the cooperative calcium binding behavior usually observed. "Unusual" fragment 1, which exhibits negative cooperativity (the type of binding behavior expected for ions interacting with a charged protein) at high concentration, also exhibit altered self-association behavior. 2. The calcium-induced spectral perturbations that are observed by fluorescence and ultraviolet difference spectroscopy are influenced by calcium-mediated dimerization. Similar spectral perturbations may also be induced by other divalent, trivalent, and monovalent ions, as well as changes in pH. Because this is a multi-site system, only limited interpretation of the spectral data is possible without calcium binding data. 3. Although strong side chain CD signals make estimation of fragment 1 secondary structure ambiguous, the CD data do indicate small changes in structure during calcium binding. Similar changes are observed upon addition of monovalent ions at high concentration or after lowering the pH. No coupling between changes in conformation and the cooperative calcium binding behavior has yet been observed to exist.

Analysis of the renaturation kinetics of bovine muscle pyruvate kinase.

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine-HCl. Subsequent dilution of the enzyme into buffer containing 2-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with half-times that vary from 185 min at 0 degrees C to 4 min at 45 degrees C. In the temperature range 0-25 degrees C, 90% of the enzymatic activity is recovered. Above about 32 degrees C, the recovery drops off sharply, wih a yield of only 13% at 45 degrees C. Removal of inactive nonspecific aggregates and denatured monomer by gel filtration yields an enzyme with the same specific activity as the starting material. At enzyme concentrations below 3 microns/mL at 16 degrees C or below 25 micron/mL at 7.8 degrees C, the reactivation kinetics show a concentration dependence. At higher concentrations of protein and at temperatures of 16 degrees C or higher, no protein concentration dependence is seen, and the rate of reactivation is described by two first-order relaxations. The rate constants have apparent activation energies of 10.6 and 11.9 kcal/mol. Combinding the results presented here with earlier work from this laboratory [Cardenas, J. M., & Dyson, R. D. (1973) J. Biol. Chem. 248, 6938-6944; Cardenas, J. M., Hubbard, D. R., & Anderson, S. (1977) Biochemistry 16, 191-197] leads to the conclusion that a rapid, major folding produces two species which undergo transconformational steps. This is followed by subunit association which yields the native tetramer.

Protein concentration dependence on aggregation behavior and properties of soybean 7S and 11S globulins during alkali-treatment.

Changes in aggregation and properties of alkali-treated soybean 7S and 11S globulins depend upon protein concentration during alkali-treatment. Such variables were investigated by viscosity, electrophoresis, circular dichroism, pulsed NMR, emulsion capacity and CaCl2 precipitation measurements. In lower protein concentrations, the intrinsic viscosity decreased and the penetrative fractions into electrophoresis gel increased. The reduced contacts of proteins during neutralization resulted in smaller aggregates. Also specific fractions which were more sensitive to protein concentration on aggregation were observed for 11S globulin. The quantity of bound water depended only on pH at 7% concentration treatment. When the gel was formed, the bound water of protein increased, e.g., 0.085 g and 0.135 g/g protein at pH 10.6 and 13.2 treatment, respectively, whereas at 1% treatment, bound water showed almost no pH dependence (about 0.13 g/g protein). Furthermore, proteins prepared at higher protein concentrations w...

Isolation and characterization of a cutinase from Fusarium roseum culmorum and its immunological comparison with cutinases from F. solani pisi

Fusarium roseum culmorum, grown on apple cutin as the sole source of carbon, was shown to produce a cutin depolymerizing enzyme. From the extracellular fluid of these F. roseum cultures, a cutinase and a nonspecific esterase were isolated utilizing Sephadex G-100, QAE-Sephadex, and SP-Sephadex chromatography. The homogeneity of the cutinase was verified by polyacrylamide disc gel electrophoresis. The molecular weight of the cutinase was estimated to be 24,300 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Electrophoretic mobility of this enzyme was between that of Cutinases I and II from Fusarium solani pisi. The F. roseum cutinase hydrolyzed p-nitrophenyl butyrate and cutin, but not p-nitrophenyl palmitate, while the nonspecific esterase hydrolyzed the long-chain esters. Amino acid composition of F. roseum cutinase was found to be similar to that of F. solani pisi Cutinase I except for differences in the number of serine, valine, and cysteine residues. The time-course, protein concentration dependence, substrate concentration dependence, and pH optimum (10.0 for cutin hydrolysis) of the F. roseum cutinase was similar to the cutinases from F. solani pisi. The F. roseum cutinase was inhibited by diisopropylfluorophosphate and paraoxon, and the [ 3H]diisopropylphosphate group was covalently attached to the enzyme upon treatment with tritiated diisopropylfluorophosphate. Therefore, it is concluded that catalysis by cutinase involves an “active serine.” Immunochemical studies with a rabbit antibody prepared against F. solani pisi Cutinase I demonstrated that Cutinase II from this organism was immunologically very similar to, but not identical to, Cutinase I. On the other hand, the cutinase from F. roseum was immunologically quite different from the cutinases isolated from F. solani pisi in that it did not cross-react with anticutinase I. However, all three cutinases were virtually identical in their sensitivity to inhibition by anticutinase I, and all three enzymes were virtually completely inhibited by the anticutinase I.

Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi

The properties of the homogeneous cutinase I, cutinase II, and the nonspecific esterase isolated from the extracellular fluid of cutin-grown Fusarium solani F. pisi (R.E. Purdy and P.E. Kolattukudy (1975), Biochemistry, preceding paper in this issue) were investigated. Using tritiated apple cutin as substrate, the two cutinases showed similar substrate concentration dependence, protein concentration dependence, time course profiles, and pH dependence profiles with optimum near 10.0. Using unlabeled cutin, the rate of dihydroxyhexadecanoic acid release from apple fruit cutin by cutinase I was determined to be 4.4 mumol per min per mg. The cutinases hydrolyzed methyl hexadecanoate, cyclohexyl hexadecanoate, and to a much lesser extent hexadecyl hexadecanoate but not 9-hexadecanoyloxyheptadecane, cholesteryl hexadecanoate, or hexadecyl cinnamate. The extent of hydrolysis of these model substrates by cutinase I was at least three times that by cutinase II. The nonspecific esterase hydrolyzed all of the above esters except hexadecyl cinnamate, and did so to a much greater extent than did the cutinases. None of the enzymes hydrolyzed alpha- or beta-glucosides of p-nitrophenol. p-Nitrophenyl esters of fatty acids from C2 through C18 were used as substrates and V's and Kms were determined...

Kinetics of Oxygen Binding to Human Hemoglobin: TEMPERATURE JUMP RELAXATION STUDIES

Abstract The kinetics of oxygen binding to human hemoglobin at pH 7, 0.1 m phosphate, 20°, 2.5 x 10-5 to 10-3 m heme has been investigated by chemical relaxation methods in order to obtain information about the elementary steps and the mechanism of the cooperative process of ligand binding. The temperature jump relaxation method allows the oxygen-binding reaction to be followed over its entire time range. The relaxation process is characterized by two phases which are well separated in time. Both the time constants and the relative amplitudes are dependent on protein and ligand concentration. The two relaxation phases can be understood qualitatively. The fast phase corresponds at low oxygen saturation mainly to the binding of the first ligand molecule and at high saturation mainly to the binding of the last ligand molecule. At intermediate oxygen saturation the fast phase is determined predominantly by the kinetics of both the first and the last step—the intermediate reaction steps do not contribute significantly. The slow relaxation phase involves all elementary binding reactions, but is determined mainly by the kinetics of the intermediate oxygen-binding steps. The relative amplitudes of the fast and the slow relaxation phases reflect strongly the population of the reaction species involved. It is concluded that hemoglobin species with one and three ligands bound must be populated measurably at equilibrium. Reaction models which assume negligible concentrations of these intermediates are therefore not valid. Three reactions have been characterized directly by their kinetic properties: (a) a fast reaction step at low oxygen saturation yielding an apparent off-rate constant of about 1000 s-1 and an apparent on-rate constant (per tetramer) of about 4 x 107 m-1 s-1; (b) a fast reaction step at high oxygen saturation, yielding an apparent on-rate constant of about 4 x 107 m-1 s-1; (c) a slow reaction, which at high oxygen concentration yields an apparent rate constant of 5 x 106 m-1 s-1. It thus appears that the binding of the first oxygen molecule to deoxyhemoglobin is a very rapid process which is characterized by an apparent rate constant as large as that found for the binding of the last oxygen molecule. The low oxygen affinity of deoxyhemoglobin arises mainly from the high dissociation rate constant, i.e. short lifetime, of Hb4O2. It cannot be decided if these kinetic properties apply equally to both the α and β chains or if only one type of chain is involved in the fast phase of the relaxation spectrum. A four-step binding model (Adair scheme) explains quantitatively the principal observations of the relaxation kinetics. Adair rate parameters have been obtained which describe the appearance of two relaxation phases only, the ligand concentration dependence (from 0 to 250 µm O2), the protein concentration dependence (from 2 x 10-5 to 10-3 m heme), and the relative and absolute relaxation amplitudes of both phases. The calculated stopped flow kinetic progress curve, using these values, is in agreement with the available data (which are in the saturation range greater than 50%) and predicts a biphasic time course, in contrast to the kinetics of CO-binding. A decision between mechanisms based upon the relaxation kinetics data can be made only with certain assumptions. The values of the Adair-recombination rate constants exclude a mechanism in which there are two very rapidly interconverting structure forms of the protein, each exhibiting equal or nearly equal intrinsic kinetic and equilibrium properties for the four subunits. A concerted mechanism might apply if structure changes are not very fast compared to ligand binding or, if the kinetic properties of the α and β chains are markedly different. However, no consistent fit of the relaxation spectrum has been obtained so far with these assumptions.

Studies on the Mechanisms of Action of Phenylalanine Hydroxylase and Its Protein Stimulator: I. ENZYME CONCENTRATION DEPENDENCE OF THE SPECIFIC ACTIVITY OF PHENYLALANINE HYDROXYLASE DUE TO A NONENZYMATIC STEP

Abstract The specific activity of phenylalanine hydroxylase from rat liver decreases markedly with increasing enzyme concentration when assayed at pH 8.0 with the naturally occurring tetrahydrobiopterin as cofactor. The decrease in specific activity is due to a sharp increase in the apparent Km for tetrahydrobiopterin at higher enzyme concentration, whereas Vmax is unchanged. This protein concentration dependence is relieved by the presence of a protein, phenylalanine hydroxylase-stimulating protein, which has been purified from rat liver (Huang, C. Y., Max, E. E., and Kaufman, S. (1973) J. Biol. Chem. 248, 4235). The stimulating protein greatly increases the apparent affinity of the hydroxylase for tetrahydrobiopterin, but has no effect on Vmax. Because of its ability to stimulate phenylalanine hydroxylase activity, the stimulating protein served as a valuable probe for the mechanism of action of the hydroxylase. Kinetic and physical studies reported here show that the dependence of specific activity on enzyme concentration is not due to the existence of different molecular weight forms of phenylalanine hydroxylase having different affinities for tetrahydrobiopterin. Neither is it due to the presence of an inhibitor which is present at a constant ratio to the enzyme. Our data are consistent with the concept that the protein concentration dependence of the specific activity of phenylalanine hydroxylase arises from the peculiar kinetic process of the enzymatic reaction. It is proposed that an intermediate is released from the enzyme during catalysis, which can recombine with the enzyme or break down nonenzymatically to product (or products). The slower nonenzymatic step (relative to the rate of recombination) imposes a limit on the over-all reaction rate, thereby resulting in an apparent enzyme concentration dependence. The proposed role of the stimulating protein is the catalysis of the nonenzymatic step. Our observations also suggest that the intermediate is either a precursor of the quinonoid dihydro form of biopterin or a complex involving biopterin, oxygen, and phenylalanine.