Spectral properties of the oestrogen-induced rat uterus peroxidase II and some of its derivatives.

1. The visible absorption spectrum of peroxidase II, isolated from the uterine tissue of oestradiol-treated rats, and some of its derivatives were recorded. The spectral properties of this enzyme are very similar to eosinophile peroxidase and lactoperoxidase, suggesting that these enzymes may have a similar form of haem as prosthetic group. 2. The uterine peroxidase is modified upon interaction with H2O2 and the difference spectrum of this modified enzyme is similar to that of complex II of lactoperoxidase. The modified enzyme was found to revert spontaneously to the native enzyme at rates which depended on the concentration of free enzyme and H2O2.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

Action of crystalline acid carboxypeptidase from Penicillium janthinellum.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.     

Protein interactions with surface-immobilized metal ions: structure-dependent variations in affinity and binding capacity with temperature and urea concentration.

We have used equilibrium binding analyses to evaluate the influence of temperature and urea on the affinity of hen egg white lysozyme and bovine pancreatic ribonuclease A for surface-immobilized Cu(II) ions. Linear Scatchard plots suggested that these model proteins were interacting with immobilized metal ions via a single class of intermediate-affinity (Kd = 10-40 microM) binding sites. Alterations in temperature had little or no effect on the immobilized Cu(II) binding capacity of either protein. Temperature effects on the interaction affinity, however, were protein-dependent and varied considerably. The affinity of lysozyme for immobilized Cu(II) ions was significantly decreased with increased temperature (0 degree C-37 degrees C), yet the affinity of ribonuclease did not vary measurably over the same temperature range. The van 't Hoff plot (1n K vs 1/T) for lysozyme suggests a straight line relationship (single mechanism) with a delta H of approximately -5.5 kcal/mol. Urea effects also varied in a protein-dependent manner. A 10-fold reduction in the affinity of lysozyme for the immobilized Cu(II) was observed with the urea concentrations up to 3 M; yet urea had no effect on the affinity of ribonuclease for the immobilized metal ions. Although the interaction capacity of lysozyme with the immobilized Cu(II) ions was decreased by 50% in 3 M urea, ribonuclease interaction capacity was not diminished in urea. Thus, temperature- and urea-dependent alterations in protein-metal ion interactions were observed for lysozyme but not ribonuclease A. The complete, yet reversible, inhibition of lysozyme- and ribonuclease-metal ion interactions by carboxyethylation with low concentrations of diethylpyrocarbonate provided direct evidence of histidyl involvement. The differential response of these proteins to the effects of temperature and urea was, therefore, interpreted based on calculated solvent-accessibilities and surface distributions of His residues, individual His residue pKa values, and specific features of the protein surface structure in the immediate environment of the surface-exposed histidyl residues. Possible interaction mechanisms involved in protein recognition of macromolecular surface-immobilized metal ions are presented.     

An Analysis of the Interaction of Sodium Dodecyl Sulfate with Lysozyme

The interaction of SDS with lysozyme was analyzed with enzyme activity and with NMR, fluorescence, and UV difference spectroscopies using various alkyl sulfates and variously modified lysozymes. SDS formed a stable complex with lysozyme without causing a gross conformational change in the enzyme molecule. Some SDS molecules bound to the active site cleft of lysozyme and therefore strongly inhibited the activity of lysozyme. Hydrophobic regions and positive charges for protein side, and a hydrophobic tail (possibly more than 8 carbons in alkyl chain) and a negative charge for detergent side were required for the formation of the complex.     

Dynamics of the Interactions of Basic Proteins with Polyribonucleotides

Pancreatic ribonuclease was irradiated in the dried state with electrons and then added to acetate buffer solutions that contained different concentrations of polyribonucleotides. Qualitatively similar results were obtained by adding a combination of unirradiated ribonuclease and lysozyme to such solutions. Such solutions scatter light strongly, and the intensity of the scattered light changes with time after mixing. The angular distribution of the scattered light was obtained as a function of time and compared with the rates at which hydrolysis products were formed. The turbidity of the solutions increases rapidly with time at the lower polyribonucleotide concentrations, and seems to result from a complex between inactive ribonuclease, or lysozyme, and oligonucleotides that appear during enzymic hydrolysis of the polynucleotides. The dissymmetry of the scattered light is approximately 5, indicating that the scattering centers are, if spherical, about 1500 A in diameter. The turbidities are remarkably high when one considers the low concentrations of protein and nucleic acid materials that are used.     

Interaction of lactoperoxidase with thiols and diiodotyrosine.

Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.     

Effect of size and charge on endocytosis of lysozyme derivatives by sinusoidal rat liver cells in vivo

Hen egg-white lysozyme has been modified by intermolecular cross-linking with dimethyl suberimidate or by acylation with acetic or succinic anhydride. Retention of the native conformation of the modified enzyme was checked by measuring enzyme activity, resistance of disulfide bridges to reduction by thiols, and susceptibility to proteases.Unmodified lysozyme and its derivatives (labelled with ¹²⁵I) were intravenously injected into nephrectomized rats, and plasma clearance and uptake by liver cells were determined. Under these conditions, about 6% of the unmodified lysozyme was taken up by liver 15 min after injection. Cross-linking led to a greatly increased uptake (up to 89% of the dose in 15 min), whereas acylation reduced the uptake to 3–4%. Cell isolations showed that the unmodified enzyme and the cross-linked derivatives were taken up by sinusoidal cells. Differential fractionation of liver homogenates indicated that the unmodified enzyme was taken up in lysosomes. The cross-linked derivatives were concentrated in the nuclear and microsomal fractions as well as in the lysosomal fraction, suggesting adsorption on plasma membranes besides uptake in lysosomes.The experiments described in this paper, together with previous results on ribonuclease and lactate dehydrogenase, indicate that endocytosis of some proteins by sinusoidal liver cells is positively correlated with size and positive charge of the molecules.     

Inhibition of two mitochondrial enzymes by gold(III) halo complexes. Evidence for a binding mechanism

Fumarase (EC 4.2.1.2) and mitochondrial L-malate dehydrogenase (EC 1.1.1.37) were both inhibited by NaAuCl₄ and KAuBr₄. The inhibition for both was measured as a function of gold complex concentration and aquation time, and the NaAuCl₄ inhibition was also measured in the presence of 0.15 M NaCl. Regeneration of the enzyme activity after NaAuCl₄ inhibition using L-cysteine, L-methionine and NaCN was also investigated. Sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis and amino acid analysis was performed on the NaAuCl₄ inhibited enzymes as well as on ribonuclease A (EC 3.1.26.2), lysozyme (EC 3.2.1.17) and liver alcohol dehydrogenase (EC 1.1.1.1). It was observed that the inhibition was proportional to the gold complex concentration but decreased markedly after aquation of the complex. In the presence of NaCl the initial rate of inactivation is essentially unaffected unless the complex has been aquated and then the initial rate is increased. Gel electrophoresis on gold complex-enzyme mixtures show polymerization for ribonuclease and lysozyme and amino acid analysis indicates that no oxidation has taken place. From these results, a binding mechanism is postulated for the inhibition of the dehydrogenases by direct displacement of a halide ligand, probably by two groups on the enzyme, at least one of which may be a sulfur containing acid.     

Downstream processing of lactoperoxidase from milk whey by involving liquid emulsion membrane

The current work deals with downstream processing of lactoperoxidase using liquid emulsion membrane from the bovine milk whey, which is a by-product from dairy industry. It is an alternate separation technique that can be used for the selective extraction of lactoperoxidase. The extraction of lactoperoxidase in liquid emulsion membrane takes place due to the electrostatic interaction between the enzyme and polar head group of reverse micellar surfactant. The optimum conditions resulted in 2.86 factor purity and activity recovery of 75.21%. Downstream processing involving liquid emulsion membrane is a potential technique for the extraction of lactoperoxidase from bovine whey.     

Spectroscopic investigations on the conformational changes of lysozyme effected by different sizes of N‐acetyl‐l‐cysteine‐capped CdTe quantum dots

The effect of N‐acetyl‐l ‐cysteine‐capped CdTe quantum dots (NAC‐CdTe QDs) with different sizes on lysozyme was investigated by isothermal titration calorimetry (ITC), enzyme activity assays, and multi‐spectroscopic methods. ITC results proved that NAC‐CdTe QDs can spontaneously bind with lysozyme and hydrophobic force plays a major role in stabilizing QDs–lysozyme complex. Multi‐spectroscopic measurements revealed that NAC‐CdTe QDs caused strong quenching of the lysozyme's fluorescence in a size‐dependent quenching manner. Moreover, the changes of secondary structure and microenvironment in lysozyme caused by the NAC‐CdTe QDs were higher with a bigger size. The results of enzyme activity assays showed that the interaction between lysozyme and NAC‐CdTe QDs inhibited the activity of lysozyme and the inhibiting effect was in a size‐dependent manner. Based on these results, we conclude that NAC‐CdTe QDs with larger particle size had a larger impact on the structure and function of lysozyme.     

Antistreptococcal Activity of Lactoperoxidase

A study of the inhibition of the growth of Streptococcus cremoris 972 by the enzyme lactoperoxidase has shown, in agreement with previous investigations, that the inhibition requires a source of both peroxide and thiocyanate. The thiocyanate may play more than one role. It stabilizes the very dilute solutions of lactoperoxidase employed in these studies, and its oxidation products may be involved in the inhibition. Binding of the enzyme by the microorganism is suggested by the fact that when the organism was preincubated with the enzyme and then in a medium free from the enzyme, but containing peroxide and thiocyanate, the growth of the organism was inhibited. This inhibition has all the properties of the enzyme-containing system. Although no dialyzable factor could be demonstrated to cause the inhibition, the inhibitory state involving peroxide, the enzyme, and thiocyanate survived for at least 60 min before cells were added to the medium. When catalase was present in the medium prior to the addition of the cells, the inhibition was completely reversed. It was only partially reversed if catalase was added a few moments after the addition of the cells. The data have been interpreted as indicating that the inhibition takes place rapidly and requires the formation of a quaternary complex of the cells, thiocyanate, peroxide, and the enzyme lactoperoxidase.     

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.     

Electric charge distribution in proteins and ionic strength dependence of reaction rate

The ionic strength dependence of the reaction rate between protein and dichloride anion radical has been investigated by flash photolysis of aqueous chloride-containing lysozyme, ribonuclease A, or insulin. The rate constant for the reaction of lysozyme or ribonuclease A with dichloride anion radicals decreases with increasing ionic strength, while it increases for insulin. The dependence was found to obey an equation derived from the theory of Debye and Hückel or the equation of Wherland and Gray for lysozyme within experimental errors. For ribonuclease A, however, it deviates largely from these equations. In the case of insulin a moderate deviation was observed. The different behavior in the ionic strength dependence is discussed in terms of the electric charge distribution in the protein molecules.     

Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry

Pressure perturbation calorimetry is a new technique that measures the heat change in a solution that results when the pressure above the solution is changed. When used in a differential calorimeter containing a dilute solution of solute in the sample cell and the corresponding buffer in the reference cell, the measured differential heat can be used to calculate the thermal coefficient of expansion of the partial volume of the solute, alpha. For proteins in dilute aqueous solution, alpha is dominated by a temperature-dependent contribution arising from the interaction of protein groups with water at the protein-solvent interface. This arises due to the effect of the protein groups on the hydrogen-bonded structure of water, and thereby clearly differentiates between structure-making hydrophobic groups and structure-breaking hydrophilic groups. This solvation contribution to alpha can be accentuated in solvents having more structure (deuterium oxide) than water and attenuated in solvents having less structure (2.8 M guanidinium sulfate). Six different proteins (chymotrypsinogen, pepsinogen, lysozyme, bovine pancreatic trypsin inhibitor, ribonuclease A, and T4 lysozyme) were examined carefully by this technique, allowing estimates of various volumetric parameters including the volume change resulting from thermal unfolding of each protein. For ribonuclease A, results obtained in both water and deuterium oxide led to an estimate of the accessible surface area of the native protein of approximately 45% relative to the fully reduced unfolded protein. Also, it was also found that ligand binding to ribonuclease A led to changes in alpha, suggesting a burial of some surface area in the ligand-protein complex.     

The succinic dehydrogenase of seedlings

Crystalline lysozyme has been interacted with an anionic, a cationic, and two nonionic surface-active agents (SAA). Quantitative precipitation of lysozyme by the ionic SAA used was obtained at ratios of the reactants consonant with the formation of stoichiometric complexes dependent upon salt linkages between the SAA and the oppositely charged groups in the enzyme. Neither of the nonionic SAA tested caused precipitation of the enzyme.The inactivation of lysozyme is shown to be constant over a 50-fold range of enzyme concentration when calculated on the basis of the ratio of SAA to enzyme. Inhibition of lysozyme activity as a result of interaction with ionic SAA was obtained only when the ionic SAA were present in substantial excess of the amount required for formation of stoichiometric complexes with oppositely charged groups in the enzyme. Neither of the two nonionic SAA studied altered the enzymatic activity of lysozyme.     

Pancreatic ribonuclease-poly G complexes: complete inhibition of poly U hydrolysis

Pancreatic ribonuclease forms large complexes with poly G in 0.1 M acetate buffer solutions (pH 5.4). These are largest when the ratio, of ribonuclease to poly G concentration, is slightly less than 2. Under the same conditions lysozyme forms still larger complexes with poly U, and these are largest when the ratio, of lysozyme to poly U concentration, is about 2.5. The ribonuclease in ribonuclease-poly G complexes digests poly U. Free ribonuclease digests the poly U in lysozyme-poly U complexes. However, when the poly G concentration is about an order of magnitude greater than that required to bind all the ribonuclease, lysozyme-bound poly U is not hydrolyzed.     

A 15N-NMR study on ribonuclease T1-guanylic acid complex

Ribonuclease T1 is highly specific for the guanylic acid residue in polyribonucleotides. To clarify the origin of the substrate specificity, the interaction sites of guanylic acid with ribonuclease T1 were investigated by the use of 15N-NMR. 95% 15N-enriched guanosine-3'-phosphate was prepared and mixed with purified ribonuclease T1. 15N-NMR spectra of the mixtures at different concentrations were obtained and compared with that of the 15N-enriched substrate alone. Upon complex formation, a 15N signal assigned to the amino group nitrogen at position 2 of guanine shifted and was significantly broadened, suggesting a strong interaction with the enzyme through the amino group. This observation is consistent with the results of studies on the substrate specificity of chemical modification. Nuclear Overhauser effects of signals assigned to N-7 and N-3 were also changed, but not shift was observed. The observations do not support the occurrence of protonation at N-7 upon complex formation, which was previously proposed.     

Increased thermal stability of proteins in the presence of naturally occurring osmolytes.

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)