The effect of viscosity on the accessibility of the single tryptophan in human serum albumin.

When reactions take place with one of the reactants tied to protein matrix, movements along the reaction coordinate towards the transition state can become coupled to structural fluctuations of the protein matrix. This investigation aims to test the assumptions underlying the arguments supporting such a coupling. A coupling is allowed only if the activation barrier is high and broad enough as shown to be the case for the proton catalyzed isotope exchange at Trp-63 of lysozyme. In the present investigation the activation barrier for the same reaction has been lowered radically in an effort to show that the coupling, as measured by the dependence of rate on solution viscosity, will diminish and ideally vanish, despite the unchanged effects of cosolvents on the chemical activities of all the reactants. The isotope exchange rate at the indole nitrogen of the single tryptophan residue of human serum albumin was measured with UV. This residue is rigidly held to the protein surface and the solvent access, although restricted, corresponds to a partially exposed residue. As a consequence, the isotope exchange rates and the bimolecular quenching rate of fluorescence by acrylamide, also measured, are high. The experiments were carried out at pH 5.2 where the molecule is in the N-form and the exchange is catalyzed by OH- ions. The activation energy of the hydroxyl catalyzed reaction is 22 kJ lower than for the proton catalyzed process. Under these conditions the exchange rate is viscosity independent both in the case of glycerol and in ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible coupling of chemical to structural dynamics in subtilisin BPN' catalyzed hydrolysis

The viscosity dependence of enzymatic catalysis was examined in subtilisin BPN' catalyzed hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-thiobenzyl ester. The viscosity of the reaction medium was varied by added glycerol, ethylene glycol, sucrose, glucose, fructose, poly(ethylene glycol) and Ficoll-400. Responses of the Michaelis-Menten parameters associated with hydrolysis were calculated from data obtained by spectrophotometric techniques. The reactions with these two substrates have catalytic rates well below the diffusion-controlled limit and thus enable us to study the viscosity effects on catalytic steps of non-transport nature. It was found that the Km values for both amide and ester reactions remained relatively independent of cosolvents. On the other hand, while the kcat values for amide were insensitive to cosolvents, those for ester were substantially attenuated except in the case of poly(ethylene glycol). The observed rate attenuations cannot be explained by changes in proton activity, water activity, dielectric constant of the reaction medium or shifts of any kinetically important pKa. Instead, the results can be adequately described by microviscosity effects on the unimolecular deacylation step with a coupling constant of 0.65 +/- 0.11. In addition, the different viscosity dependence in the acylation vs deacylation step can be rationalized in terms of fluctuation-dependent chemical dynamics of proton transfers in the context of the Bogris-Hynes model.     

The coupling of catalytically relevant conformational fluctuations in subtilisin BPN' to solution viscosity revealed by hydrogen isotope exchange and inhibitor binding

We have measured the tritium outexchange of subtilisin BPN'. A consistent and rather small group of hydrogens was isolated by their sensitivity to inhibitor binding. The viscosity dependence of exchange from these inhibitor protected hydrogens was then examined in 0.05 M MES buffer, pH 6.5 and 10 degrees C. The viscosity of the reaction medium was varied by added glycerol and ethylene glycol. The exchange rates were corrected to be compared at identical hydroxyl ion and water activity. The salient observation is the strikingly similar viscosity coupling behavior when compared to the deacylation step of ester hydrolysis catalyzed by the same enzyme (Ng and Rosenberg, Biophysical Chemistry, 39 (1991) 57). We have obtained a viscosity coupling constant of 0.68 -/+ 0.18 for hydrogen exchange in glycerol (cf. 0.65 -/+ 0.11 for deacylation in glycerol, sucrose, glucose and fructose); 1.67 -/+ 0.07 for outexchange (cf. 1.92 -/+ 0.09 for deacylation), in the presence of ethylene glycol. The two reactions are very chemically dissimilar, yet they show very similar viscosity coupling behavior. This together with the well established role of structural fluctuations in hydrogen exchange implies a similar role of structural fluctuations in the deacylation step of subtilisin BPN' catalyzed ester hydrolysis.     

pH dependence of individual tryptophan N-1 hydrogen exchange rates in lysozyme and its chemically modified derivatives

Nuclear magnetic resonance analyses have been made of the individual hydrogen-deuterium exchange rates of tryptophan indole N-1 hydrogens in native lysozyme and its chemically modified derivatives including lysozyme with an ester cross-linkage between Glu-35 and Trp-108, lysozyme with an internal amide cross-linking between the epsilon-amino group of Lys-13 and the alpha-carboxyl group of Leu-129, and lysozyme with the beta-aspartyl sequence at Asp-101. The pH dependence curves of the exchange rates for Trp-63 and Trp-108 are different from those expected for tryptophan. The pH dependence curve for Trp-108 exchange exhibits the effects from molecular aggregation at pH above 5 and from a transition between the two conformational fluctuations at around pH 4. The exchange rates for tryptophan residues in native lysozyme and modified derivatives are not correlated with the thermodynamic or kinetic parameters in protein denaturation, suggesting that the fluctuations responsible for the exchange are not global ones. The exchange rates for tryptophan residues remote from the modification site are perturbed. Such tryptophan residues are found to be involved in a small but distinct conformational change due to the modification. Therefore, the perturbations of the N-1 hydrogen exchange rates are related to the minor change in local conformation or in conformational strain induced by the chemical modification.     

Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin.

Protein-ligand binding and enzyme activity have been shown to be regulated by solvent viscosity, induced by the addition of viscous cosolvents. This was indirectly interpreted as an effect on protein dynamics. However, viscous cosolvents might affect dynamic, e.g., viscosity, as well as thermodynamic properties of the solution, e.g., activity of solution components. This work was undertaken to examine the effect of viscous cosolvent on the structural dynamics of proteins and its correlation with dynamic and thermodynamic solution properties. For this purpose we studied the effect of viscous cosolvent on the specific ultrasonic absorption, delta mu, of bovine serum albumin, at pH = 7.0 and at 21 degrees C, and frequency range of 3-4 MHz. Ultrasonic absorption (UA) directly probes protein dynamics related to energy dissipation processes. It was found that the addition of sucrose, glycerol, or ethylene glycol increased the BSA delta mu. This increase correlates well with the solvent viscosity, but not with the cosolvent mass concentration, activity of the solvent components, dielectric constant, or the hydration of charged groups. On the grounds of these results and previously reported findings, as well as theoretical considerations, we propose the following mechanism for the solvent viscosity effect on the protein structural fluctuations, reflected in the UA: increased solvent viscosity alters the frequency spectrum of the polypeptide chain movements; attenuating the fast (small amplitude) movements, and enhancing the slow (large amplitude) ones. This modulates the interaction strength between the polypeptide and water species that "lubricates" the chain's movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.     

Contribution of water to free energy of hydrolysis of pyrophosphate

The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products.     

Protein folding stabilizing time measurement: a direct folding process and three-dimensional random walk simulation

Protein particles undergo Brownian motion and collisions in solution. The diffusive collisions may lead to aggregation. For proteins to fold successfully the process has to occur quickly and before significant collision takes place. The speed of protein folding was deduced by studying the correlation time of a lysozyme refolding process from autocorrelation function analysis of the mean collision time and aggregation/soluble ratio of protein. It is a measure of time before which an aggregate can be formed and also is the time measure for a protein to fold into a stable state. We report on the protein folding stabilizing time of a lysozyme system to be 25.5-27.5 micros (<+/-4%) between 295 and 279K via direct folding experimental studies, supported by a three-dimensional random walk simulation of diffusion-limited aggregation model. Aggregation is suppressed when the protein is folded to a stable form. Spontaneous folding and diffusion-limited aggregation are antagonistic in nature. Meanwhile, the resultant aggresome, suggested by Raman and mass spectroscopy, may be formed by cross-linkages of disulfide bonds and hydrophobic interactions.     

Solvent interactions with the active site of the pig heart cytosolic aspartate aminotransferase.

Aqueous solvent interactions with the chromophoric pyridoxal phosphate prosthetic group of aspartate aminotransferase (EC 2.6.1.1) were analyzed quantitatively with ethylene glycol, glycerol, dimethylsulfoxide (DMSO), sucrose, and xylitol as cosolvents. The smaller cosolvents perturb the visible absorption and visible dichroic spectra of the free enzyme, but this solvent perturbation is not observed with the acidic enzymeglutarate complex. Addition of cosolvents caused an increase in the enzyme's affinity for glutarate. This increase in affinity resulted from an increase in the acidic dissociation constant (pK₂) of the enzyme-glutarate complex. The changes in the acidic dissociation constant of the enzyme-glutarate complex, upon addition of cosolvents, correlate well with the changes observed in the pK_a's of carboxylic acids in comparable solvents. Since these solvents have little effect on the pK_a of the enzyme itself, it is concluded that the increase in affinity is due to a specific solvation effect on a carboxyl group of the enzymebound glutarate, rather than resulting from a conformational change in the protein.     

Structural Stability of Myoglobin in Organic Media

The structural stability of metmyoglobin in organic solvents and cosolvents was investigated aiming the choice of a suitable medium to perform its dissolution with maintenance of the native folding. The spectroscopic behavior of metmyoglobin solution in UV–Visible and circular dichroism was used to evaluate the solubility and the secondary structure. The results were dependable of the chemical structure of the organic compounds, their polarity and content, in the case of cosolvents. Protic solvents showed better ability than the aprotic ones for the biomolecule dissolution, since they are able to establish hydrogen bonds. Solvents with high polarity usually damage the secondary structure of the protein. Myoglobin was dissolved in pure methanol, ethylene glycol and glycerol. The secondary structure was retained in some extent. The controlled addition of sodium dodecyl sulfate to myoglobin aqueous solution changed the surface moiety of the protein. The complex was extracted to hexane with efficiency of 77%.     

Raman spectroscopic characterization of tryptophan side chains in lysozyme bound to inhibitors: role of the hydrophobic box in the enzymatic function.

The state of H-bonding and the hydrophobic interaction of six tryptophan side chains in lysozyme bound to substrate-analogous inhibitors were investigated by combining H----D exchange labeling and Raman difference spectroscopy. The frequency of the W17 band due to Trp-63 shifts downward upon inhibitor binding, indicating a specific and strong H-bond formation between the N1 site of the side chain and the inhibitor molecule. On the other hand, the H-bonding state of Trp-62 in the complex is as weak as that in inhibitor-free lysozyme, suggesting no contribution of this residue to the inhibitor binding. Intensity increases of W17 and W18 bands observed upon inhibitor binding are, respectively, ascribed to an increase at Trp-28 and a decrease at Trp-111 in hydrophobic interactions with the environment. The environmental changes are explained consistently by a movement of the Met-105 side chain sandwiched by two indole rings of Trp-28 and 111 in the direction from Trp-111 to Trp-28. The sandwich structure in a core domain, hydrophobic box, and its rearrangement are considered to play an important role in the enzymatic function of lysozyme.     

Elongation of actin filaments is a diffusion-limited reaction at the barbed end and is accelerated by inert macromolecules

We used a fluorescence method to measure the rate constants for the elongation of pyrene-labeled actin filaments in a number of different solvents. The absolute values of the rate constants were established by electron microscopy. Using glycerol, sucrose, or ethylene glycol to vary the solution viscosity, the association rate constant (k+) was 10(7) M-1 s-1 viscosity-1 (in centipoise). Consequently, plots of 1/k+ versus viscosity are linear and extrapolate to near the origin as expected for a diffusion-limited reaction where the rate constant approaches infinity at zero viscosity. By electron microscopy, we found that this inhibitory effect of glycerol is almost entirely at the fast growing, barbed end. For the pointed end, plots of 1/k+ versus viscosity extrapolate to a maximum rate of about 10(6) M-1 s-1 at zero viscosity, so that elongation at the pointed is not limited by diffusion. In contrast to these small molecules, polyethylene glycol, dextran, and ovalbumin all cause a concentration (and therefore viscosity)-dependent increase in k+. At any given viscosity, their effects are similar to each other. For example, at 3 centipoise, k+ = 2.2 X 10(7) M-1 s-1. We presume that this is due to an excluded volume effect that causes an increase in the thermodynamic activity of the actin. If the proteins in the cytoplasmic matrix have a similar effect, the association reactions of actin in cells may be much faster than expected from experiments done in dilute buffers.     

The inverse relationship between protein dynamics and thermal stability

Protein powders that are dehydrated or mixed with a glassy compound are known to have improved thermal stability. We present elastic and quasielastic neutron scattering measurements of the global dynamics of lysozyme and ribonuclease A powders. In the absence of solvation water, both protein powders exhibit largely harmonic motions on the timescale of the measurements. Upon partial hydration, quasielastic scattering indicative of relaxational processes appears at sufficiently high temperature. When the scattering spectrum are analyzed with the Kohlrausch-Williams-Watts formalism, the exponent beta decreases with increasing temperature, suggesting that multiple relaxation modes are emerging. When lysozyme was mixed with glycerol, its beta values were higher than the hydrated sample at comparable temperatures, reflecting the viscosity and stabilizing effects of glycerol.     

The influence of glycerol on hydrogen isotope exchange in lysozyme

Hydrogen isotope exchange rates for lysozyme in glycerol cosolvent mixtures [D. G. Knox and A. Rosenberg (1980) Biopolymers 19 , 1049–1068] have been analyzed as functions of solvent viscosity and glycerol activity in an attempt to determine which solvent properties influence protein internal dynamics. The effect of glycerol on the fast- and slow-exchanging protons is different. Slow-exchanging protons [H(t) < 20] are slowed by ever-increasing amounts as H(t) decreases. However, comparison with data for the effect of glycerol on the thermal unfolding of lysozyme [K. Gekko (1982) J. Biochem. 19 , 1197–1204] indicates that the large decrease in exchange rates for the slow protons is not consistent with a local unfolding mechanism of exchange. These effects are also too large to be easily rationalized in terms of solvent viscosity. Instead, we suggest that the large effect of glycerol on exchange of the slow protons is due to a “compression” of the protein, as a result of thermodynamically unfavorable interactions of glycerol with the protein surface. This reduces the protein void volume, which in turn decreases the probability of conformational transitions required for exchange of the slowest protons. Present data do not allow a distinction to be made between thermodynamic (glycerol activity) and dynamic (solvent viscosity) influences on exchange rates for the fast-exchanging protons, although the effect of glycerol on these protons is also probably too large to be consistent with a local unfolding mechanism. In this case, glycerol decreases the rate of catalyst diffusion within the protein matrix, either by decreasing the probabilities or amplitudes of “gating” reactions that allow passage of the catalyst from the solvent to the exchange site, or by increasing the relaxation times for these conformational rearrangements.     

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat‐induced deamidation and β‐elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alchol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S‐carboxy‐methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers––PEG and PVP––and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution. Biotechnol. Bioeng. 2012; 109: 2543–2552. © 2012 Wiley Periodicals, Inc.     

Viscosity dependence of O2 escape from respiratory proteins as a function of cosolvent molecular weight.

Laser photodissociation of respiratory proteins is followed by fast geminate recombination competing with escape of the oxygen molecule into the solvent. The escape rate from myoglobin or hemerythrin has been shown previously to exhibit a reciprocal power-law dependence on viscosity. We have reinvestigated oxygen escape from hemerythrin using a number of viscous cosolvents of varying molecular weight, from glycerol to dextrans up to 500 kDa. In isoviscous solutions, the strong viscosity dependence observed with small cosolvents is progressively reduced upon increasing the cosolvent's molecular weight and disappears at molecular weights greater than about 100 kDa. Thus, viscosity is not a suitable independent parameter to describe the data. The power of the viscosity dependence of the rate coefficient is shown here to be a function of the cosolvent's molecular weight, suggesting that local protein-solvent interactions rather than bulky viscosity are affecting protein dynamics.     

Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering

The dynamics of lysozyme in the picosecond timescale has been studied when it is in dry and hydrated powder form and when it is embedded in glycerol, glycerol–water, glucose and glucose–water matrices. The investigation has been undertaken through elastic neutron scattering technique on the backscattering spectrometer IN13. The dynamics of dry powder and embedded-in-glucose lysozyme can be considered purely vibrational up to 100 K, where the onset of an anharmonic contribution takes place. This contribution can be attributed to the activation of methyl group reorientations and is described with an Arrhenius trend. An additional source of anharmonic dynamics appears at higher temperatures for lysozyme in hydrated powders and embedded in glycerol, glycerol–water and glucose–water matrices. This second process, also represented with an Arrhenius trend, corresponds to the so-called protein dynamical transition. Both the temperature where such a transition takes place and the magnitude of the protein mean square displacements depend on the environment. The dynamical response of the protein to temperature is put in relationship with its thermal stability.     

The association reaction of yeast alcohol dehydrogenase with coenzyme is partly diffusion-controlled in solvents of increased viscosity

The steady-state kinetics of the yeast and liver alcohol dehydrogenase catalyzed reduction of aldehydes were examined in solvent mixtures of increased viscosity. This was done to investigate the effects of diffusion control on the fast association of NADH with the enzymes. Both glycerol and sucrose were unsatisfactory as viscosogens, as they inhibited the enzyme, but poly(ethylene glycol)/water mixtures were satisfactory. The 5-fold faster reaction of yeast alcohol dehydrogenase with NADH is partly diffusion controlled, whereas the slower liver alcohol dehydrogenase reaction showed no diffusion effects. These results are consistent with a yeast alcohol dehydrogenase active site that has relatively little steric hindrance to NADH binding. It is estimated that contributions to this association reaction from diffusion control and chemical activation control are equal at a solvent viscosity of 10 cP. Thus, under physiological conditions of increased viscocity the NADH association may be significantly affected by diffusion effects. In order to estimate accurately the maximum diffusion-controlled rate constant from diffusion theory, the diffusion coefficients of NADH were measured in poly(ethylene glycol)/water mixtures and were found to vary inversely as the solvent viscosity raised to the power of 0.5. The non-Stokesian behaviour of molecules as large as NADH in polymer/water mixtures may be a serious limitation to the routine use of poly(ethylene glycol) as a viscosogen for diffusion studies.     

An Apparatus for the Concentration of Large Volumes of Dilute Protein Solutions to A Predetermined Volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.     

An apparatus for the concentration of large volumes of dilute protein solutions to a predetermined volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5l) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.