Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions.

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre-crystallization conditions in NaCl-containing media were recorded at scattering angles from 20 degrees to 90 degrees. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3-fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.     

Determination of second virial coefficient of proteins using a dual-detector cell for simultaneous measurement of scattered light intensity and concentration in SEC-HPLC

A method is proposed for the measurement of the B22 value of proteins in aqueous solutions in flow-mode that utilizes a novel fabricated dual-detector cell, which simultaneously measures protein concentration and the corresponding scattered light intensity at 90 degrees , after the protein elutes from a size-exclusion column. Each data point on the chromatograms obtained from the light scattering detector and the concentration (ultraviolet) detector is converted to Rayleigh's ratio, Rtheta, and concentration, c, respectively. The B22 value is calculated from the slope of the Debye plot (Kc/Rtheta versus c) generated from a range of concentrations obtained from these chromatograms for a single protein injection. It is shown that this method provides reliable determination of the B22 values for such proteins as lysozyme, chymotrypsinogen, and chymotrypsin in various solution conditions that agree well with those reported in literature.     

The Complexing of Lysozyme with Poly C and Other homopolymers

Lysozyme forms very large complexes with poly C, in acetate buffer solutions (pH 5.4), when the ratio of lysozyme to poly C concentration is 3/2. When this is less than 3/8 there is virtually no complexing, as evidenced by the low light-scattering power of such mixtures. At such relatively high poly C concentrations, the addition of pancreatic ribonuclease causes both the intensity and dissymmetry of scattering to rise to very high values after which time the intensity falls exponentially with time and with very little change in dissymmetry. Other homopolymers also form largest complexes with lysozyme at characteristic concentration ratios.     

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.     

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.     

The evaluation of the temperature effect and hydrolysis conditions for amino acid analysis

A systematic investigation of the optimal temperature and hydrolysis time for amino acid analysis has been carried out under various conditions. It is found that some simplification and increase in speed relative to the conventional protocol of employing vacuum-sealed tubes and 110 C/24-72 hour hydrolysis can be achieved without loss of accuracy and performance in amino acid analyses of proteins and peptides. The effects of hydrolysis temperature and heating time on the recoveries of various labile and hydrophobic amino acids are exemplified in the hydrolysis of oxidized ribonuclease A, lysozyme and lens crystallin. The method provides a rapid processing of multiple samples within hours instead of days with the potential for the total automation of amino acid analysis starting from the preparation of protein hydrolysates.     

Protein purification with vapor-phase carbon dioxide

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.     

Toxic properties of the cell wall of gram-positive bacteria

The biological activity of Odontomyces viscosus, which has been reported to cause periodontal disease in hamsters, was examined. The microorganism was cultured anaerobically in Brain Heart Infusion broth, and the cells were harvested. The washed cells were injected intradermally into the abdomen of rabbits. After 72 hr, a well-defined, firm, raised nodule (about 1.0 by 1.5 cm) with an erythematous border was seen at the injection site. Suspensions of cell wall and cytoplasmic material were injected intradermally, and the lesions appeared only at the site of cell wall injection. The cell walls, which were then treated with trypsin, pepsin, and ribonuclease, again produced the characteristic lesion. These nodular dermal lesions persisted for a minimal time of 10 days. The enzymatically treated cell walls were then hydrolyzed with 1 n HCl, and such hydrolysis up to 1 hr failed to alter the toxic activity of the cell walls. Similar dermal nodular lesions were obtained by injection of enzymatically treated cell walls of strains of Staphylococcus aureus, Streptococcus groups B, C, E, F, K, Lactobacillus casei, and Actinomyces israelii. Treatment with hot and cold trichloroacetic acid solutions and proteolytic enzymes, or with formamide, yielded insoluble fractions which produced the characteristic nodular lesions. The size of the lesion resulting from injection of these fractions was proportional to the amount of the injected material. The active fraction, which does not appear susceptible to hydrolysis by lysozyme, is thought to be cell wall mucopeptide. Histological studies showed skin abscesses due to the toxic reaction; however, in addition to the acute inflammatory reaction, there was local eosinophilia.     

The reaction of penicillin with proteins.

The mode of reaction of benzylpenicillin with two proteins was studied, with particular reference to the allergenicity of penicillin. These reactions, with pig insulin, and with hen's-egg-white lysozyme, were carried out in neutral solution at 37 degrees C. High concentrations of penicillin are needed to label the proteins, owing to concurrent hydrolysis of penicillin. Evidence has been obtained that the penicillin-reactive sites on the insulin molecule are the alpha-amino group at the N-terminus of the A chain and the epsilon-amino group of the lysine residue; whereas a site of reaction with lysozyme appears to be the epsilon-amino group of lysine-116.     

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.     

Preparation of allosteric ribonuclease.

A method for the preparation of allosteric ribonuclease from bovine pancreas is described. The effects of freeze-drying ribonuclease from acid and alkaline solutions on plots of velocity versus substrate concentration for the hydrolysis of 2':3'-cyclic CMP are examined. Comparison of these plots with the plots obtained with severeal commercial enzyme preparations indicates that the conformation of the enzyme is dependent on the method of preparation. Aging experiments demonstrate that further conformational changes occur at different rates, depending on the methods of storage. Results suggest that the allosteric behaviour of ribonuclease has not always been observed with commercial preparations, owing to variations in methods of preparation and storage of the enzyme.     

Characterization of the Extracellular Ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

Characterization of the extracellular ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

Probing lysozyme conformation with light reveals a new folding intermediate

A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a greater degree of lysozyme swelling than UV light, thus allowing for the protein conformation to be controlled with light.     

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)     

Effect of nucleic-acid-binding compounds on the hydrolytic activity of various ribonucleases.

Studies were conducted on the stimulatory effect that various nucleic-acid-binding compounds have on the hydrolysis of RNA and polyribonucleotides by pancreatic ribonuclease A and by other ribonucleases. The stimulatory activity of chloroquine on tRNA hydrolysis by pancreatic ribonuclease was due to the formation of oligonucleotides of a wide range of sizes and was not due to the formation of very short ( n greater than 5) oligonucleotide fragments of tRNA. The dextrorotatory and levorotatory isomers of chloroquine did not differ in their ability to stimulate the hydrolysis of tRNA by pancreatic ribonuclease A. In addition to chloroquine and primaquine, other nucleic-acid-binding compounds (e.g., quinacrine, lucanthone, and proflavin) stimulated the hydrolysis of tRNA by pancreatic ribonuclease A. Chloroquine did not alter the rate of hydrolysis by pancreatic ribonuclease A of low-molecular-weight substrates (cytidine cyclic 2':o'-monophosphate, uridine cyclic 2':3'-monophosphate, cytidylyl-adenosine, or uridylyl-uridine). Furthermore, chloroquine and primaquine did not affect the hydrolysis of poly(A) by high concentrations of pancreatic ribonuclease A. In studies on the hydrolysis of tRNA by other endoribonucleases, several of the nucleic-acid-binding compounds (e.g., quinacrine and ethidium) exhibited appreciable inhibition of both ribonuclease N1 and ribonuclease T1. None of the compounds tested stimulated the activity of ribonuclease T1, and only chloroquine, and perhaps lucanthone, stimulated the hydrolysis of tRNA by ribonuclease N1.     

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available. Received: 8 August 1996 / Accepted: 18 October 1996     

Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.     

Lysozyme crystal growth, as observed by small angle X-ray scattering, proceeds without crystallization intermediates

A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles. Received: 28 July 1997 / Revised version: 4 December 1997 / Accepted: 5 December 1997     

Effects of cross-linked dimers of ribonuclease A or of lysozyme on the processing of endocytosed peroxidase by hepatoma cells.

Cross-linked dimers of ribonuclease, added at a concentration of 0.05 mg/ml to the culture medium of hepatoma (HTC) cells, were previously shown to inhibit intracellular degradation of peroxidase taken up by endocytosis. Intracellular localization showed that endocytosed peroxidase does not reach lysosomes in dimer-treated cells. The present study shows that preloading of lysosomes with fluorescent anti-peroxidase IgG, obtained by exposing HTC cells for 48 h to 0.1 mg of antibody/ml, restores intracellular degradation of endocytosed peroxidase. Moreover, accumulation of peroxidase into lysosomes, which no longer occurs in dimer-treated cells, occurs again under these conditions. We conclude that inhibition of transfer of peroxidase from phagosomes to lysosomes is most likely to be the alteration resulting from the exposure of the cells to ribonuclease dimer, rather than inhibition of fusion between phagosomes and lysosomes. The dimer of another basic protein, lysozyme added at a concentration of 0.2 mg/ml to the culture medium, is shown to induce the same type of effects as does the dimer of ribonuclease; the half-life of endocytosed peroxidase increased from 5 to 15 h after 2 h exposure of HTC cells to dimerized lysozyme. The effect of both dimers on intracellular protein processing can be reversed by addition of 100 mm-galactose to the culture medium, up to 5 h after pretreatment of the cells. The dimers of ribonuclease A or of lysozyme have thus probably the same mechanism of action. Evidence that the two dimers share the same binding sites on the cells is presented.