Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.     

Stabilizing effect of amphiphilic excipients on the freeze-thawing and freeze-drying of lactate dehydrogenase

The effects of amphiphilic excipients on the inactivation of lactate dehydrogenase (LDH) during freeze-thawing and freeze-drying were studied. Some amphiphilic excipients such as hydroxypropyl-beta-cyclodextrin (HP-beta-CD), CHAPS, polyethylene glycol (PEG) 3350, and sucrose fatty acid monoester prevented LDH inactivation during freeze-thawing and freeze-drying at a lower concentration than sugars and amino acids. Polyoxyethylene 9 lauryl ether and PEG 400 protected LDH during freeze-thawing but not during freeze-drying. The buffer concentration of the solution to be freeze-dried (10, 50, and 200 mM) affected the stabilizing effect of trehalose, but not that of HP-beta-CD. (c) 1994 John Wiley & Sons, Inc.     

Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels]

The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.     

Characteristics of proteinaceous additives in stabilizing enzymes during freeze-thawing and -drying

Protein-stabilizing characteristics of sixteen proteins during freeze-thawing and freeze-drying were investigated. Five enzymes, each with different instabilities against freezing and dehydration, were employed as the protein to be stabilized. Proteinaceous additives generally resulted in greater enzyme stabilization during freeze-thawing than sugars while the degree of stabilization for basic lysozyme and protamine were inferior to that of neutral and acidic proteins. Freeze-drying-induced inactivation of enzyme was also reduced by the presence of a proteinaceous additive, the extent of which was lower than that for a sugar. In both freeze thawing and freeze drying, the enzymes stabilization by the proteinaceous additive increased with increasing additive concentration. The enhancement of enzyme inactivation caused by pH change was also reduced in the presence of proteinaceous additives. The combined use of a sugar such as sucrose and dextran tended to increase the stabilizing effect of the proteinaceous additive.     

Glucosylglycerol and glucosylglycerate as enzyme stabilizers

Compatible solutes constitute a diverse class of low-molecular-mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like α, α-trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R-2-O-α-D -mannopyranosyl-glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high-temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2-O-α-D -glucopyranosyl-sn-glycerol) and glucosylglycerate (R-2-O-α-D -glucopyranosyl-glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of α,α-trehalose. Glucosylglycerate in concentrations of ≥0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. α,α-Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.     

Protein fractions of the ova, embryos and skeletal muscles of the loach affecting the stability and activity of lactate dehydrogenase

When lactate dehydrogenase obtained from Misgurnus muscles and purified to the homogeneous state is incubated for 16 hat 38 degrees C, its activity lowers down to 10% of the initial value. Extracts of egg cells, embryos or skeletal muscles of the mentioned fish species added to the enzyme solution decrease considerably its inactivation. Proteins stabilizing the activity of lactate dehydrogenase are revealed in the supernatant liquid obtained after salting out of the above extracts with 60% sulphate ammonium saturation. These proteins are in fractions with the molecular weight below 45 kDa. Among proteins with the molecular weight 10 kDa there are polypeptides which exert an activation effect on lactate dehydrogenase. This effect is intensified with the presence of insulin.     

Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui.

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.     

Thermal stability of lactate dehydrogenase in the complex with the anion polyelectrolyte poly(styrene sulfonate)

The temperature stability of the cytoplasmic enzyme of the glycolysis of lactate dehydrogenase from a pig muscle (isoenzyme M4) in a complex with the anion polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, the own protein fluorescence, and circular dichroism. Calorimetric investigations of complex of lactate dehydrogenase with poly(styrenesulphonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulphonate)/lactate dehydrogenase, though at 20 degrees C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulphonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Temperature stability of lactate dehydrogenase in complex with anionic polyelectrolyte poly(styrenesulfonate)

The temperature stability of the cytoplasmic enzyme of glycolysis, lactate dehydrogenase from pig muscle (isoenzyme M4) in complex with anionic polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, own protein fluorescence, and circular dichroism. Calorimetric investigations of the complex of lactate dehydrogenase with poly(styrenesulfonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulfonate)/lactate dehydrogenase, though at 20°C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulfonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Intrinsic versus extrinsic stabilization of enzymes: the interaction of solutes and temperature on A4-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes.

We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis), which has lower values for kcat and the Michaelis constant for pyruvate ( K m PYR), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (Tm(APP)). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in Tm(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A4-LDH was decreased most by solutes and increased most by temperature. In contrast, the K m PYR values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A4-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A4-LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A4-LDH orthologs may evolve independently.     

Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants.

To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals. Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits. They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide. Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms. However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride. When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB. These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C. However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained. The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.     

Counteraction of urea destabilization of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes

Intracellular fluids of marine elasmobranchs (sharks, skates and rays), holocephalans and the coelacanth contain urea at concentrations averaging 0.4m, high enough to significantly affect the structural and functional properties of many proteins. Also present in the cells of these fishes are a family of methylamine compounds, largely trimethylamine N-oxide with some betaine and sarcosine, and certain free amino acids, mainly beta-alanine and taurine, whose total concentration is approx. 0.2m. These methylamine compounds and amino acids have been found to be effective stabilizers of protein structure, and, at a 1:2 molar concentration ratio of these compounds to urea, perturbations of protein structure by urea are largely or fully offset. These counteracting effects of solutes on proteins are seen for: (1) thermal stability of protein secondary and tertiary structure (bovine ribonuclease); (2) the rate and extent of enzyme renaturation after acid denaturation (rabbit and shark lactate dehydrogenases); and (3) the reactivity of thiol groups of an enzyme (bovine glutamate dehydrogenase). Attaining osmotic equilibrium with seawater by these fishes has thus involved the selective accumulation of certain nitrogenous metabolites that individually have significant effects on protein structure, but that have virtually no net effects on proteins when these solutes are present at elasmobranch physiological concentrations. These experiments indicate that evolutionary changes in intracellular solute compositions as well as in protein amino acid sequences can have important roles in intracellular protein function.     

Time-dependent effects of trimethylamine-N-oxide/urea on lactate dehydrogenase activity: an unexplored dimension of the adaptation paradigm.

Given that enzymes in urea-rich cells are believed to be just as sensitive to urea effects as enzymes in non-urea-rich cells, it is argued that time-dependent inactivation of enzymes by urea could become a factor of overriding importance in the biology of urea-rich cells. Time-independent parameters (e.g. Tm, k(cat), and Km) involving protein stability and enzyme function have generally been the focus of inquiries into the efficacy of naturally occurring osmolytes like trimethylamine-N-oxide (TMAO), to offset the deleterious effects of urea on the intracellular proteins in the urea-rich cells of elasmobranchs. However, using urea concentrations found in urea-rich cells of elasmobranches, we have found time-dependent effects on lactate dehydrogenase activity which indicate that TMAO plays the important biological role of slowing urea-induced dissociation of multimeric intracellular proteins. TMAO greatly diminishes the rate of lactate dehydrogenase dissociation and affords significant protection of the enzyme against urea-induced time-dependent inactivation. The effects of TMAO on enzyme inactivation by urea adds a temporal dimension that is an important part of the biology of the adaptation paradigm.     

Preferential binding of two compatible solutes to the glycan moieties of Peniophora lycii phytase

Regulation of hydration behavior, and the concomitant effects on solubility and other properties, has been suggested as a main function of protein glycosylation. In this work, we have studied the hydration of the heavily glycosylated Peniophora lycii phytase in solutions (0.15-1.1 m) of the two compatible solutes glycerol and sorbitol. Osmometric measurements showed that glycerol preferentially binds to phytase (i.e., glycerol-glycoprotein interactions are more favorable than water-glycoprotein interactions resulting in a preferential accumulation of glycerol near the protein interface), while sorbitol is preferentially excluded from the hydration sphere (water-glycoprotein interactions are the more favorable). To assess contributions from carbohydrate and peptide moieties, respectively, we compared phytase (Phy) and a modified, yet enzymatically active form (dgPhy) in which 90% of the glycans had been removed. This revealed that both polyols showed a pronounced and approximately equal degree of preferential binding to the carbohydrate moiety. This preferential binding of polyols to glycans is in contrast to the exclusion from peptide interfaces observed here (for dgPhy) and in numerous previous reports on nonglycosylated proteins. Despite the distinct differences between peptide and carbohydrate groups, glycosylation had no effect on the stabilizing action provided by glycerol and sorbitol. On the basis of this, it was concluded that the carbohydrate mantle of Phy is equally accessible in the native and thermally denatured states, respectively (most likely fully accessible in both), and thus that its interactions with compatible solutes have little or no effect on conformational equilibria of the glycoprotein. For solubility and aggregation equilibria, on the other hand, the results suggest a polyol-induced stabilization of monomeric forms.     

Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects

Arginine has been used to suppress protein aggregation and protein-protein or protein-surface interactions during protein refolding and purification. While its biotechnology applications are gradually expanding, the mechanism of these effects of arginine has not been fully elucidated. Arginine is more effective at higher concentrations, an indication of weak interactions with the proteins. The effects of weakly interacting additives, such as arginine, on protein solubility, stability and aggregation have been explained from three different approaches: i.e., (1) the effects of additives on the structure of water, (2) the interactions of additives with the amino acid side chains and peptide bonds and (3) the preferential interactions of additives with the proteins. Here we have examined these properties of arginine and compared with those of other additives, e.g., guanidine hydrochloride (GdnHCl) and certain amino acids and amines. GdnHCl is a strong salting-in agent and denatures proteins, while betaine is a protein stabilizer. Several amino acids and amine compounds, including betaine, which stabilize the proteins, are strongly excluded; i.e., the proteins are preferentially hydrated in these solutions. On the other hand, GdnHCl preferentially binds to the proteins. Arginine is intermediate between these two extreme cases and shows a more complicated pattern of interactions with the proteins. The effects of additives on water structure, e.g., the surface tension of aqueous solution of the additives and the solubility of amino acids in the presence of additives also shed light on the mechanism of the effects of the additives on protein aggregation. While arginine increases the surface tension of water, it favorably interacts with most amino acid side chains and the peptide bonds, a property shared with GdnHCl. Thus, we propose that while arginine is similar to GdnHCl in the amino acid level, arginine interacts with the proteins differently from GdnHCl.     

Generation of multilamellar and unilamellar phospholipid vesicles

Multilamellar and unilamellar vesicles can be generated by a variety of techniques which lead to systems with differing lamellarity, size, trapped volume and solute distribution. The straight-forward hydration of lipid to produce multilamellar vesicles (MLVs) results in systems which exhibit low trapped volumes and where solutes contained in the aqueous buffer are partially excluded from the MLV interior. Large trapped volumes and equilibrium solute distributions can be achieved by freeze-thawing or by ‘reverse phase’ procedures where the lipid is hydrated after being solubilized in organic solvent. Unilamellar vesicles can be produced directly from MLVs by extrusion or sonication or, alternatively, can be obtained by reverse phase or detergent removal procedures. The advantages and limitations of these techniques are discussed.     

Role of hydrophobicity in the binding of coenzymes. Appendix. Translational and rotational contribution to the free energy of dissociation.

We calculate the loss of surface area accessible to solvent associated with coenzyme binding in Clostridium flavodoxin, in dogfish lactate dehydrogenase, and in lobster glyceraldehyde-3-phosphate dehydrogenase. The coenzymes are nearly buried in the complexes and lose on the order of 600 A2, while the proteins lose a similar amount of accessible surface area. Some of the loss can be attributed to conformation changes in the protein, at least in the case of lactate dehydrogenase, where we show that the apoenzyme has a larger accessible surface area than the holoenzyme. Using known correlations with the hydrophobic contribution to the free energy, we demonstrate that hydrophobicity is the major source of stabilization free energy in FMN binding to flavodoxin and in NAD binding to the two dehydrogenases: it contributes 25 to 30 kcal/mol to the free energy of dissociation, more than required in order to compensate for the loss of six degrees of translational/rotational freedom by the coenzyme.     

The electrostatic contribution to interactions of some enzymes with polyelectrolytes

To explain the inhibitory action of polyelectrolytes on enzymes and, in particular, to define potentially reactive zones for the binding of polyelectrolyte, the electric potential of enzymes lactate dehydrogenase and glutamate dehydrogenase was calculated using the solution of the Poisson-Boltzmann equation by a numerical method with the use of the Gauss-Seidel relaxation method at three pH values: 6.5, 7.0, and 8.0 and three values of ionic strength: 50, 100, and 150 mm. On the basis of these calculations and their visualization, representative sites for favorable binding of polyanions were determined as extended areas on the surface of proteins with the positive potential in the neutral pH region. It was shown that there is a correlation between the area of positive potential and the efficiency of enzyme inactivation for a number of pH values and concentrations of salt for two enzymes. The calculations performed allowed one to explain the inhibitory action of polyelectrolytes on the specified enzymes to understand the difference between the values of polyelectrolyte inactivation constants for the two enzymes and estimate the minimal areas of the positive potential on the protein surface that provide their effective inhibition.     

Effects of fluid shear on immobilized enzyme kinetics.

There have been a number of reports concerning the damaging effects of shear on globular proteins in solution. Some recent work has indicated, however, that globular proteins in solution are relatively stable, but may be inactivated at air-liquid interfaces during shearing. This study investigated the effects of fluid shear on immobilized enzyme activity. Immobilized enzyme reactors were built to operate with the enzyme immobilized at the boundary of a fluid flow field. Two different enzymes, penicillinase and lactate dehydrogenase, were covalently bound to the interior surface of nylon tubes. Fluid shear rate was changed by varying the flow rate of substrate (reactant) solution through the tube, and fluid shear stresses were increased by increasing the viscosity of the recirculating solution. There were no observed effects of fluid shear on immobilized penicillinase or lactate dehydrogenase activity at shear rates of up to 10,350 s-1 or at shear stresses of up to 73 Pa.     

Effects of thermodynamic nonideality on protein interactions. Equivalence of interpretations based on excluded volume and preferential solvation

Published results on the stabilization of proteins by sucrose (J.C. Lee and S.N. Timasheff, J. Biol. Chem. 256 (1981) 7193) have been reexamined and interpreted in terms of thermodynamic nonideality. The composition dependence of activity coefficients may be accounted for on a statistical-mechanical basis using the concept of excluded volume. An expression is derived in which the effect of sucrose on determination of the partial specific volume of a protein, previously interpreted in terms of preferential protein solvation, is also seen to be attributable to excluded volume. Gel chromatographic studies of the reversible unfolding of alpha-chymotrypsin are presented which demonstrate temperature- and sucrose-mediated changes in the effective volume of the enzyme. These measurements support the quantitative interpretation of the stabilization in terms of thermodynamic nonideality arising from the difference between covolumes for sucrose and the two isomeric states of alpha-chymotrypsin. By establishing the equivalence of the two approaches that have been used to account for the effects of inert solutes on protein transitions, the present investigation eliminates the need for any distinction between such solutes on the basis of molecular size; and also enhances greatly the potential sensitivity of thermodynamic nonideality as a means of probing protein isomerizations, since greater displacement of the equilibrium position may be effected by small rather than by macromolecular solutes present at the same weight concentrations.