Investigations of protein–solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid-dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 m). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid-inactivated porcine heart lactate dehydrogenase reflects a non-native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non-native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid-dissociated porcine heart lactate dehydrogenase as well as other viscosity-dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.
In order to accommodate environmental water stress (e.g. salinity, desiccation, freezing), many organisms accumulate one or more osmotically active solutes (osmolytes) . Two classes of osmolytes are recognized. Those that stabilize proteins in vitro without significantly perturbing protein function are defined as compatible osmolytes . Counteracting osmolytes, such as trimethylamine oxide (TMAO), tend to buffer proteins and other cellular constituents against elevated concentrations of chaotropic agents such as urea [1,3,4].
Efforts to delineate the molecular basis of osmolyte action have generated large amounts of literature on protein–solute interactions. Virtually all studies of the effects of osmolytes on protein stability have demonstrated that these chemical chaperones strongly favor the native conformation. For example, TMAO protects ribonuclease T1 against thermal denaturation . There are also several reports by Bolen and coworkers showing that both proline and TMAO, as well as other osmolytes, have a propensity for ‘forcing’ intrinsically unstable polypeptides to fold into more compact, native-like, conformations (e.g. ).
Interest in protein–osmolyte interactions arises from several considerations. Inasmuch as unfolded polypeptides would be expected to be particularly sensitive to environmental stress and protease action, it is reasonable to ask whether osmolytes may facilitate the folding of nascent polypeptides; i.e. perhaps one of the functions of osmolytes is to act as chemical chaperones during the terminal stages of protein synthesis. Observations showing the effects of osmolytes on protein conformation in vivo provide support for this hypothesis [6–10].
Several investigators have noted that some osmolytes are of potential practical use in the rescue of inclusion bodies [e.g. 7,11]. It is also conceivable that coexpression of appropriate osmolytes may retard or prevent the formation of such aggregates in expression systems.
With apparently few exceptions [e.g. 1,7,11–14], previous investigations did not include testing of the possible effects of osmolytes on the kinetics of reactivation of denatured polypeptides. Also, study of the effects of osmolytes on the reactivation of oligomeric, cytosolic proteins seems to have been somewhat limited (see Discussion).
In the light of these considerations, we chose to explore the possible effects of osmolytes on renaturation/reassociation of lactate dehydrogenase (LDH; EC 22.214.171.124). The choice of experimental system was based on the following considerations. LDH is an oligomer, comprised of four polypeptides of identical size, and its refolding and reactivation following denaturation/dissociation in various solvent media have been extensively investigated. Thus, the pathway for refolding and reassociation is generally well established . Reactivation of LDH following denaturation in 6 m guanidine hydrochloride (GdnHCl) begins with the fully unfolded polypeptide subunits and the time course reflects a complex series of molecular events that include folding, dimerization of monomers and association of the dimers to form the active tetramer (see Discussion). However, when acid-dissociated monomers are stabilized by sodium sulfate, the rate limiting step is restricted to association of the dimer to produce the active tetrameric species . Thus, study of renaturation (following unfolding in GdnHCl), as well as reassociation (following inactivation at low pH), allowed exploration of the effect of osmolytes on the reactivation of the enzyme from two very different conformational states.
In this investigation we explored the effects of representative compatible and counteracting osmolytes on the kinetics and extent of reactivation of LDH from beef heart following denaturation in 6 m GdnHCl, as well as following dissociation at pH 2.3 in the presence of sodium sulfate. In contrast with expectation, based on results obtained with other proteins [5,11,16], TMAO and proline were found to inhibit both the time course and extent of renaturation of LDH from bovine heart (BHLDH) following unfolding by the chaotropic agent, as well as reactivation of the enzyme following inactivation at low pH. Reactivation of acid-dissociated LDH from porcine heart (PHLDH) was also sensitive to both osmolytes. Evidence was obtained that the molecular species that is enzymatically active during the initial stages of reactivation of acid-inactivated PHLDH reflects an altered conformation and that this non-native species is kinetically stabilized by interaction with osmolytes.
Materials and methods
Dithiothreitol, ethylenediaminetetraacetic acid (EDTA), GdnHCl, LDH from bovine heart (type III), nicotinamide adenine dinucleotide (reduced, NADH), sodium pyruvate and Tris base were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lactate dehydrogenase from porcine heart was from Roche Molecular Biochemical (Indianapolis, IN, USA). l-Proline was from Sigma-Aldrich (Sigma Ultra) or Fluka (MicroSelect; Milwaukee, WI, USA). Trimethylamine N-oxide dihydrate (> 99%) was from Fluka. N,N-Bis(hydroxyethyl)-2-aminoethane sulfonic acid (Bes) was from Research Organics (Cleveland, OH, USA).
Enzyme stock solutions
Stock solutions of enzyme (≈ 5–9 mg·mL−1) were prepared by dialysis (≈ 5 °C) against 100 mm Tris/HCl, 1 mm EDTA (pH 7.4; prior to denaturation in GdnHCl) or 100 mm sodium phosphate, 100 mm EDTA, 1 mm dithiothreitol (pH 7.6; prior to dissociation at low pH).
Enzyme concentration (mg protein·mL−1) was calculated from = 1.5 for BHLDH  and 1.4 for PHLDH . The preparation of LDH from beef heart was composed of approximately 70% H4 and 30% H3M ; i.e. > 92% H subunits. The preparation from pig heart was composed of approximately 95% H4 and a small fraction of H3M; it contained ≈ 98% H subunits.
Assay of enzymatic activity
This was performed at room temperature (21–24 °C) by measurement of the rate of decrease in absorbance at 340 nm with a Shimadzu 1601PC spectrophotometer. Reaction mixtures (1.021 mL, in polystyrene cuvettes) contained 128 µm NADH, 350 µm sodium pyruvate (unless stated otherwise) and approximately 1 pmol enzyme (added last). The buffer for the assay was 100 mm potassium Bes (or Tris/HCl, pH 7.0, for enzyme denatured in GdnHCl) or 100 mm sodium phosphate, 1 mm EDTA (pH 7.6, for enzyme inactivated at acid pH). The specific activities of the BHLDH and PHLDH were 317 and 310 U·mg−1, respectively, at 22 °C. Molar concentration of enzyme was based on a tetramer molecular mass of 140 000 Da.
None of the osmolytes tested inhibited enzymatic activity of the untreated enzyme at the concentrations that were present during enzyme assays (≤ 60 mm TMAO, ≤ 150 mm proline; data not shown).
Denaturation and acid-induced dissociation
Apart from where indicated, unfolding in 6 m GdnHCl was initiated by the addition of a 10 µL aliquot of stock enzyme (containing 76–81 µg LDH) to 90 µL 6.7 m GdnHCl in 100 mm Tris/HCl , 1 mm EDTA (pH 7.4). The inactivation mixtures were incubated for 10 min at room temperature. Inactivation at pH 2.3 was initiated by addition of 8–9 µL LDH (containing 52–54 µg protein) to 91–92 µL cold 100 mm sodium phosphate, 800 mm sodium sulfate (pH 2.3); samples were incubated on ice for 60 min. All incubations, for both inactivation and reactivation, were performed in polypropylene tubes.
Renaturation following unfolding in GdnHCl was initiated by 50-fold dilution in 100 mm Tris/HCl, 1 mm EDTA, 2 mm dithiothreitol (pH 7.4), with or without the indicated concentration of the specified osmolytes (proline or TMAO); stock solutions of osmolytes were adjusted to pH 7.4. All reactivations were performed at room temperature. Protein concentration in these reactivation mixtures was typically 15–16 µg·mL−1.
Reactivation of acid-inactivated enzyme was initiated by 50- or 100-fold dilution in 100 mm sodium phosphate, 1 mm EDTA, 1 mm dithiothreitol (pH 7.6) plus or minus the indicated osmolytes (TMAO or proline) at room temperature; stock solutions of osmolytes were adjusted to pH 7.6.
The protein concentrations in reactivation mixtures are specified in the relevant figure legends. Aliquots were removed from reactivation mixtures at the indicated times after initiation of reactivation and assayed for enzymatic activity as described above.
Molecular graphics analysis
Two models of the structure of the H4 isoform of LDH are found in the RCSB Protein Data Bank (PDB). The PDB code for porcine H4 is 5LDH. Analysis of both this model and the one for the major isoform from human cardiac muscle (PDB code 1I0Z) by deep view[20,21] shows that the latter is the superior model. This assessment was based on the fact that opening up the model for 5LDH in deep view, reveals a lengthy list of missing amino acid sidechains; this reflects uninterpretable electron density in those areas. There are no such uncertainties in the model for 1I0Z. As the primary structures for LDH H4 from pig and human heart are 95% identical (97% similar) we chose to base our analysis of buried and surface residues on the human enzyme. The model for 1I0Z is for the dimer. We obtained a model for the tetramer from 1I0Z through protein explorer, using the link to protein quaternary analysis (PQS ).
Effect of osmolytes on the reactivation of bovine LDH following denaturation in GdnHCl
Proline inhibits the rate of reactivation. The equilibrium level of reactivation of BHLDH in the absence of osmolytes, following denaturation in 6 m GdnHCl (65 ± 2.8%, relative to the activity of the untreated enzyme; data not shown), was several-fold greater than that reported for similar studies of PHLDH under similar conditions . For each experiment, the activity for the control (no osmolyte in the reactivation mixture), determined 24 h after initiation of reactivation, was taken as representing the equilibrium level of reactivation under the experimental conditions employed and was assigned a value of 1.0. The enzymatic activity observed at intermediate times (with or without osmolyte) was expressed as a fraction of this equilibrium control value and was defined as ‘relative reactivation’. The initial time course for reactivation of controls was routinely hyperbolic (Fig. 1).
The kinetic profile for reactivation in the presence of 1 m proline was virtually indistinguishable from that of controls, but in the presence of increasing concentrations of proline, the time course became increasingly sigmoidal (Fig. 1). Due to this sigmoidicity, the effects of intermediate levels of proline (2 m and 3 m) were most pronounced during the early phase of reactivation; e.g. while inhibition by 2 m proline was significant during the first hour, by 5 h the activity approached that of controls. Relative reactivation in the presence of 4 and 5 m proline remained at less than 0.1 throughout the indicated time period.
Inhibition of the extent of reactivation of LDH by proline is correlated with the unusual solution properties of proline. Relative reactivation, based on activity determined at presumed equilibrium (24 h after initiation of reactivation), was taken as a measure of the extent of reactivation. The effect of proline concentration on the extent of reactivation of LDH is summarized in Fig. 2A. At 1 m proline, reactivation was unaffected, and 2 m proline diminished the extent of reactivation only slightly, but inhibition was progressively more significant above 3 m; at 5 m proline, inhibition was virtually complete.
The concentration dependence of the viscosity of aqueous solutions of proline is somewhat unusual relative to that of compounds of similar molecular weight ; values for the viscosities of proline solutions over the concentration range from 1 to 6 m are included in Fig. 2. Inhibition of reactivation is most pronounced in solutions of proline that exhibit the greatest viscosity. This relationship is illustrated more clearly in Fig. 2B.
Trimethylamine oxide is a potent inhibitor of the extent of reactivation of GdnHCl-denatured LDH. The effect of TMAO on the relative reactivation at equilibrium is summarized in Fig. 3. The data for proline are included for comparison. The counteracting osmolyte, TMAO, was the more potent inhibitor of reactivation. The concentration of TMAO required to reduce the relative reactivation at equilibrium to 0.5 was approximately 700 mm, while the concentration of proline that was required to elicit a similar level of inhibition was 3.2 m.
Perturbation of the reactivation of acid-dissociated LDH by proline and TMAO
Reactivation of acid-dissociated BHLDH was inhibited by proline. Both the initial rate and extent of reactivation of bovine LDH, following dissociation at pH 2.3, were significantly greater than for enzyme denatured in GdnHCl. Similar differences have been reported by Jaenicke and coworkers in studies of the porcine LDH . The time required for relative reactivation in the absence of osmolytes to reach 0.5 during reactivation from GdnHCl was ≈ 30 min (Fig. 1), but ≈ 5 min for enzyme inactivated at acid pH in the absence of the chaotropic agent (Fig. 4A). Activity at apparent equilibrium in the absence of osmolytes following acid dissociation approached 90% of the activity of the untreated enzyme (data not shown). The time courses for reactivation of acid-dissociated enzyme in the absence of osmolytes, as well as in the presence of 1 or 2 m proline, were hyperbolic and virtually indistinguishable (Fig. 4A); 3 m proline, however, was inhibitory and the time required to attain a relative reactivation of 0.5 was increased to ≈ 20 min. At 4 m proline the time course became slightly sigmoidal and the time required to reach a relative reactivation of 0.5 was ≈ 90 min (Fig. 4A); the initial rate of reactivation in the presence of 5 m proline was virtually zero and relative reactivation rose only slightly (to ≈ 0.1) over the 5 h incubation period (Fig. 4A).
Proline concentrations up to 3 m did not inhibit the extent of reactivation (Fig. 4B); at 4 and 5 m proline, relative reactivation was reduced to approximately 0.85 and 0.3, respectively. A limited analysis of the effect of high proline concentrations on the reactivation of acid-inactivated PHLDH yielded similar results (data not shown).
Trimethylamine oxide inhibits the rate of reactivation of bovine LDH following inactivation at acid pH. The time required to reach a relative reactivation of 0.5 was increased from ≈ 5 to ≈ 25 min by 1 m TMAO; at 5 h, relative reactivation with the osmolyte approached that of controls (Fig. 5A). When reactivation was performed in the presence of 2 m TMAO, relative reactivation was less than 0.1 at 5 h after initiation of reactivation.
TMAO (1 m) enhances the initial rate of reactivation of porcine LDH following inactivation at pH 2.3, while 2mTMAO inhibits reactivation. The time course for reactivation of acid-dissociated PHLDH in the absence of osmolyte was hyperbolic (Fig. 5B), and reactivation reached apparent equilibrium at approximately 90% of the activity of the untreated enzyme (data not shown). The initial rate of reactivation was slower that that of the beef enzyme; the time required to reach a relative reactivation of 0.5 was increased from ≈ 5 min (Fig. 5A, BHLDH) to ≈ 20 min (Fig. 5B, PHLDH). In marked contrast to the inhibitory effect of 1 m TMAO on reactivation of the bovine enzyme (Fig. 5A), this concentration of the osmolyte enhanced the rate of reactivation of the pig dehydrogenase; the time required to reach a relative reactivation of 0.5 was reduced from ≈ 20 min to ≈ 7 min (Fig. 5B). At 5 h after initiation of reactivation the activities of controls and the mixture containing 1 m TMAO reached similar levels. In the presence of 2 m TMAO, however, the initial rate of reactivation of PHLDH was strongly inhibited, but slightly less so than with BHLDH (Fig. 5A,B).
TMAO (2 m) significantly reduced the extent of reactivation of both bovine and porcine LDH. In the presence of 1 m TMAO, the relative reactivation at presumed equilibrium for the porcine enzyme was slightly greater than that of controls, while the value for the bovine enzyme was reduced to ≈ 0.9 (Fig. 6). In 2 m TMAO, the relative reactivation at equilibrium was reduced to ≈ 0.4 and ≈ 0.1 for PHLDH and BHLDH, respectively.
As with reactivation of enzyme denatured in GdnHCl, TMAO was a more potent inhibitor of reactivation of acid-denatured BHLDH than proline. The concentration of TMAO required to reduce relative reactivation at equilibrium to 0.5 for enzyme dissociated at pH 2.3 was approximately 1.5 m (Fig. 6), whereas for a similar level of inhibition by proline, the concentration required was approximately threefold greater (compare Figs 4B and 6).
The time course of reactivation of acid-dissociated LDH in the presence of proline is dependent on protein concentration. The initial time course for the reactivation of PHLDH in the absence of osmolytes was dependent on the concentration of LDH protein in the reactivation mixture (Fig. 7A), consistent with a pathway involving a rate-determining association step (see Discussion). If attenuation, by osmolytes, of the reactivation of enzyme inactivated at low pH involves modulation of a rate-determining association step, the kinetics of reactivation in the presence of an inhibitory concentration of osmolyte should also be dependent on protein concentration. Proline-inhibited reactivation of acid-dissociated LDH was also protein concentration-dependent (Fig. 7B). TMAO-inhibited reactivation of acid-dissociated enzyme, however, was independent of protein concentration (Fig. 7C).
Analysis of interfacial contacts in lactate dehydrogenase from cardiac muscle. The program ms was used to calculate the surface area buried in each subunit upon formation of the tetramer, based on the coordinates provided in PDB file 1I0Z, as modified as described in Materials and methods. Approximately 55% of these buried sidechains are nonpolar in nature (Table 1). When the model for the native tetramer was analyzed for groups on the surface that are exposed to solvent using deep view[20,21], ≈ 43% were found to be nonpolar (data not shown).
Table 1. Analysis of interfacial contacts in lactate dehydrogenase from cardiac muscle. The program MS  was used to calculate the surface buried in each subunit upon formation of the tetramer, based on the coordinates provided in PDB file 1I0Z, as modified as described in Materials and methods. For these calculations, a probe radius of 1.7 Å was used.
Surface area (Å2) by residue class
Aromatic (nonpolar): F
Aromatic (polar): W,Y
Total area represented by sidechains
Area represented by hydrophobic sidechains
1852 Å2 (54.8%)
Area represented by polar sidechains
1526 Å2 (45.2%)
Effect of osmolytes on the kinetic properties of PHLDH during reactivation following acid-induced dissociation. The H4 isoform of LDH is particularly sensitive to pyruvic acid . An early study of the reactivation of LDH from avian cardiac muscle, following unfolding in GdnHCl, showed that during the initial stage of reactivation there were one or more enzymatically active species that exhibited diminished thermal stability and reduced inhibition by pyruvic acid . It was of interest therefore to determine whether during reactivation of acid-inactivated PHLDH there were enzymatically active species that exhibited altered pyruvate sensitivity and whether concentrations of proline and/or TMAO that inhibited reactivation kinetically stabilized these non-native molecular species.
Two identical aliquots of PHLDH were inactivated at pH 2.3; during reactivation, one reactivation mixture was assayed at 350 µm pyruvate and the other at 10 mm pyruvate. The ratio of the rate observed at the lower pyruvate concentration to that at the higher substrate concentration for the untreated enzyme was typically ≈ 2.6 (data not shown). For reactivating enzyme, however, the activity observed at 10 mm pyruvate during the initial stages of reactivation was slightly higher than that with 350 µm pyruvate; in the absence of osmolyte, at approximately 2 min after initiation of reactivation, the enzymatic rates became equivalent (Fig. 8A). Subsequently, the rate observed at the lower substrate concentration became increasingly greater than that at the higher substrate concentration. The addition of the osmolytes to the reactivation mixtures markedly increased the period during which the enzymatically active species was less sensitive to substrate inhibition. In the presence of proline (Fig. 8B; 3.4 m) or TMAO (Fig. 8C; 1.6 m), the activity at the higher substrate concentration remained greater than that at the lower substrate concentration until approximately 20 and 10 min in the presence of proline and TMAO, respectively. At presumed equilibrium (24 h after initiation of reactivation) the ratio of the rate observed at the lower substrate concentration to that at the higher substrate concentration was the same (within 5%) as for the untreated enzyme (data not shown).
The results that are summarized in Fig. 8 represent a typical experiment. Three such experiments were performed. While there was significant variation in absolute values for points that determine the time courses, all the patterns were similar to those shown in Fig. 8. This experimental variation probably reflects the complexity of the molecular events associated with the generation of the putative non-native intermediate and its conversion to the native conformation, together with interaction with the osmolytes. It is significant, however, that the large differences between the controls (no osmolyte in the reactivation mixture) and experimental (with proline or TMAO in the reactivation mixtures) samples in the time required for the rates observed at the lower and higher substrate concentrations to become equivalent were similar among experiments. The results for these experiments are summarized in Table 2.
Table 2. Effect of proline and TMAO on kinetic properties of PHLDH following inactivation at pH 2.3. Enzyme was inactivated and reactivated with and without the indicated concentration of osmolytes, and assays of enzymatic activity were performed at 350 µm and 10 mm pyruvic acid as described in the legend for Fig. 8. The rates determined at 350 µm and 10 mm were designated as L and H, respectively. Numbers in parentheses indicate the number of independent determinations.
Time at L/H = 1.0
2.3 ± 0.5 min (4)
3.4 m Proline
16.7 ± 3.1 min (3)
1.6 m TMAO
12.5 ± 2.3 min (3)
Early in the development of concepts regarding the interplay between the effects on protein structure and function of perturbants, such as urea, and counteracting osmolytes (such as TMAO and alkyl amines), it was recognized that alone, the latter might be harmful . Studies of the levels and distribution of counteracting osmolytes among various organisms support this hypothesis. The concentrations of alkyl amines (mostly TMAO) in muscles of several deep-sea organisms approach 300 mmol·kg tissue−1, but are elevated only in species in which a perturbant is also present ; TMAO is high in deep-sea animals where pressure is a perturbant, as well as in all cartilaginous fishes where urea is a perturbant. It was also demonstrated that several stabilizing solutes enhance the formation of abnormal amyloid structures in vitro.
The data summarized in Fig. 3 seem to be consistent with this hypothesis. While the reduction in relative reactivation at equilibrium (following denaturation in GdnHCl) by 250 mm TMAO was modest, it was significant. This concentration of the osmolyte approaches the physiological range for some organisms. Thus, to the extent that refolding and reassociation of the polypeptides of LDH following denaturation in GdnHCl mimic the folding of the nascent protein, TMAO may be a physiologically significant regulator of protein folding in some deep-sea organisms. Perhaps shallow-water organisms accumulate less TMAO because it would interfere with protein folding. Possible further support for this hypothesis is provided by the observations indicating that osmolytes may sometimes stabilize altered protein conformations during folding (Fig. 8, and see below).
The effects of proline on folding and reassociation of LDH described here occur over a concentration range that is much higher than estimates of the level of accumulation of proline in various organisms under physiological conditions.
It is likely that molecular chaperones are involved in the folding of LDH in vivo, but results of such investigations have not been reported. Studies of the interplay among nascent (or unfolded) polypeptides, molecular chaperones and osmolytes seem to be limited. An investigation of the effects of salt and heat stresses on aggregation and disaggregation of malate dehydrogenase showed that several osmolytes modulate the effects of complex chaperone networks on protein folding . In vitro studies showed that physiological levels of trehalose stabilized an inactive, partially folded, conformation of luciferase and inhibited chaperone-assisted reactivation of luciferase that had been unfolded in GdnHCl [7,8].
We are aware of only two prior reports of the effect of osmolytes on the reactivation of denatured LDH. An early study by Yancey and Somero  showed that following inactivation at low pH, TMAO (200 mm) enhanced both the rate and extent of reactivation of the somewhat unstable isoform of LDH from rabbit muscle. In addition to the species and isoform differences, those experiments differed in two significant respects from the current study; dissociation was performed in the absence of sodium sulfate to stabilize the monomers, and reactivation mixtures contained 1.5 mm NAD+. The results were somewhat similar to the enhanced rate of reactivation of acid-dissociated PHLDH by 1 m TMAO (Fig. 5B). There are apparently no other reports of the effects of either of the osmolytes employed in this investigation on the reactivation of lactate dehydrogenase; however, glycerol was shown to retard the rate of reactivation of acid-dissociated porcine LDH isoforms . These reports appear to be the first recorded instances of the effects of osmolytes on the renaturation/reactivation of an oligomeric, cytosolic protein.
In assessing possible molecular bases of the observations described in this communication, it is useful to consider some of what is known about (a) the kinetics and mechanism of refolding and reactivation of LDH following denaturation/dissociation in various media; (b) the energetics of differential interactions of solvent and osmolytes with sidechains and the polypeptide backbone; (c) the anomalous colligative properties of proline in aqueous solution; and (d) the effects of proline and TMAO on the stability, folding and biological activity of LDH, as well as a few other proteins.
Studies of the time course of folding of several tetrameric enzymes, following denaturation in various media have led to the following general pathway for folding and association [15,27]:
where m represents the fully unfolded monomeric polypeptide and M* represents the partially folded monomer having significant secondary structure, while M designates the monomeric polypeptide having assumed its tertiary structure; D and T indicate the dimer and tetramer, respectively. Thus, the model includes the major molecular species in the transition from random coil to native tetramer. Inasmuch as the investigations by Jaenicke and coworkers have provided strong support for the proposition that only the tetramer is enzymatically active, appearance of activity parallels the formation of native structure (see below, however).
For the H4 and M4 isoforms of LDH from porcine heart and muscle, the equilibrium constant for the 4M to 2D conversion is of the order of 108 L·mol−1, and the rate approaches that for a diffusion controlled reaction; the slow, first order 4M* to 4M conversion is preceded by a very fast 4m to 4M* transition that occurs before the initial measurement is performed .
The time course of reactivation following acid-induced dissociation in the presence of sodium sulfate reflects a somewhat simpler sequence of molecular events than that following unfolding in the presence of a chaotropic agent. In this instance, the first and second order events in the mechanism of renaturation are uncoupled; reactivation begins with structured monomers. Following the rapid equilibrium of the diffusion-controlled association of monomers to form the dimer, the rate determining step is the association of dimers to form the active tetramer; under these conditions the kinetic profile is second order and hyperbolic. Experimental support for this reassociation pathway was provided by studies of the porcine LDH isoforms by Jaenicke and coworkers [15,27,36].
There have been no similar dissociation/reactivation studies of LDH from bovine tissues, but given the structural and functional similarities among the major isoforms of LDH from heart tissue of various species , and the similarity in kinetic profiles for reactivation, in the absence of osmolyte, of acid-inactivated PHLDH and BHLDH observed in this study (Fig. 5), it is probable that the mechanism proposed for reassociation of the porcine dehydrogenase also applies to the bovine enzyme. There is a clear difference, however, in the effect of TMAO concentration on reactivation. While 2 m osmolyte inhibits reactivation of both enzymes, 1 m TMAO enhances the initial rate of reactivation of the porcine dehydrogenase but inhibits initial stages in the reactivation of the bovine enzyme (Fig. 5). This most likely reflects species differences in sensitivity of exposed residues to interaction with the solute (see below), due to conformational variations arising from differences in primary structure.
Useful insights regarding differential interactions of sidechains and the polypeptide backbone with osmolytes were provided in a recent review by Bolen and Baskakov . Analysis of the free energy of transfer of the sidechains and polypeptide backbone of ribonuclease T1 from water to osmolyte showed that interactions between osmolyte and sidechains were uniformly favorable (negative ΔG) but interactions between osmolyte and the polypeptide backbone were unfavorable (positive ΔG). For both the native and denatured conformations, the magnitude of the unfavorable interaction with the polypeptide backbone was much greater than the favorable interaction with the sidechains. The principal difference for the two conformations was that the magnitude of the free energy change for transfer of the backbone of the denatured conformation from water to osmolyte solution was much greater than that for the native conformer. The net result of this solvophobic effect, which they term ‘osmophobic’, is the stabilization of the native conformation. Their analysis further showed that although proline is similar to TMAO as a stabilizing solute, on a molar basis, it is significantly less effective. Interaction of both proline and TMAO with sidechains of amino acids is uniformly favorable, and while both osmolytes interact more strongly with polar residues, interaction of these residues with proline is significantly stronger than with TMAO .
The protein concentration dependence of the effect of proline on reactivation of PHLDH, following inactivation at pH 2.3 (Fig. 7B), is consistent with inhibition of an association process, and with the hypothesis that reactivation in the presence of the osmolyte follows a path similar to that in buffer alone. The perturbation of reactivation of acid-dissociated LDH by this osmolyte may be partially mediated by interactions between proline and sidechains of amino acid residues. Such interactions could arise from clustering of sidechains that lie at the interfaces of folded monomers or dimers that are involved in the stabilization of quaternary structure, as in the formation of dimers and/or the enzymatically active tetramer. To the extent that osmolytes bind preferentially to interfacial domains of monomers or dimers, and/or a non-native conformation of the presumed tetramer (see below), formation of the fully native LDH tetramer would be retarded.
As noted above, analysis of the buried surface area for each subunit in the LDH tetramer showed that these interfacial regions are approximately 55% nonpolar (Table 1), while approximately 57% of those on the surface of the fully native tetramer that are exposed to solvent were found to be polar (see Results). Thus, there is not a differential clustering of polar residues (with which proline and TMAO interact preferentially ) in the regions that interact to form the tetramer. It is conceivable that the strength of the interaction of the osmolytes with sidechains of certain residues (or some combinations of them) is greater than the interaction with others and that these residues are distributed preferentially in the interfacial regions.
Efforts to explain the effects of high concentrations of proline on refolding and/or reassociation of LDH must also include consideration of the unusual colligative properties of this osmolyte [11,16,25,26,39]. It is unusually soluble, and unlike most low molecular weight compounds, the relative viscosity of aqueous proline solutions increases exponentially with increasing concentration; the rise is particularly dramatic above 3.5 m ( and Fig. 2).
The rates of second order processes, such as the rate-determining association of dimers to generate active tetramers in the reactivation of acid-dissociated LDH (see above), are inversely proportional to the viscosity of the medium. It should also be noted that if there are motions on the surface of a monomer, which are large enough to affect the monomer–monomer (or dimer–dimer) interface, then they could be viscosity- dependent, irrespective of the diffusion of the monomer (or dimer) per se. The correlation between the effect of increasing proline concentration on viscosity and on reactivation of acid-dissociated enzyme (Figs 2 and 4B) supports the proposition that much of the effect of proline on reactivation following dissociation at low pH is due to the high viscosity of the medium. The viscosity of glycerol solutions undoubtedly contributed to the inhibition of reactivation of acid-inactivated LDH that was previously reported .
It was suggested that some of the unusual colligative properties of proline in aqueous solution are due to its association to form multimeric species, the size of which is concentration-dependent . The structure proposed for these supramolecular assemblies remains somewhat speculative [11,16], but it is plausible that some of the effects of proline on reassociation following acid dissociation (or renaturation from GdnHCl) involve association of polypeptide intermediates in the reactivation pathway with these postulated multimeric proline species. It is likely that the energetics of interaction between exposed sidechains on the surface of intermediates in the folding/reassociation pathway and these proline assemblies differ significantly from their interaction with proline monomers.
Trimethylamine oxide is a more potent inhibitor of reactivation of acid-dissociated enzyme than proline; e.g. while 2 m proline had virtually no effect on the level of reactivation at presumed equilibrium, 2 m TMAO inhibited the extent of reactivation > 50% (≈ 60% for PHLDH and ≈ 90% for BHLDH; Fig. 6). As noted above, evidence from studies by others supports the hypothesis that in the presence of sodium sulfate, the acid-dissociated subunits are stabilized in their native conformation . However, the absence of protein concentration dependence on inhibition of reactivation of acid-dissociated enzyme by TMAO (Fig. 7C) indicates that, unlike proline, inhibition of reactivation by this compound is not due to attenuation of a rate–determining association step. It is also very unlikely that the effect of TMAO includes a viscosity component, but it is probable that this osmolyte inhibits reactivation by stabilization of non-native conformation(s) of one or more intermediates in the reactivation pathway, presumably by favorable interaction between TMAO and exposed clustered sidechains. Perhaps the sodium sulfate-stabilized monomers are in equilibrium with a partially folded monomer (non-native) that is stabilized by binding of exposed residues to TMAO.
While the major isoforms of LDH in skeletal muscle (M4) and cardiac tissue (H4) are very similar, there are very significant differences. For example, H4 is typically more stable than M4, and is much more sensitive to inhibition by pyruvic acid [17,29,37,40].
Analysis of substrate inhibition provided additional insight regarding the basis of osmolyte effects on the time course of reactivation of acid-inactivated PHLDH. As outlined above, one interpretation of the inhibitory effects of osmolytes on the initial rate of reactivation of acid-dissociated lactate dehydrogenase suggests that proline and TMAO may stabilize one or more intermediates in the reactivation pathway. Although previous studies have shown that the tetramer is the only enzymatically active molecular species during the reactivation of lactate dehydrogenase [15,27], in the course of the current studies, it was found that during the early stages of the reactivation of PHLDH following acid-induced inactivation, the enzymatically active species exhibits a kinetic property (i.e. diminished substrate inhibition) that differs markedly from that of the untreated enzyme or reactivated enzyme at presumed equilibrium (see above). The presence of inhibitory concentrations of osmolytes during reactivation of acid-inactivated PHLDH prolonged the lifetime of one or more enzymatically active (presumably tetrameric) molecular species that was/were less sensitive to pyruvate inhibition approximately five- to sevenfold (Fig. 8 and Table 2). These observations are consistent with the proposition that the molecular species that is (are) enzymatically active during the initial period of reactivation has (have) an altered conformation(s) and that concentrations of proline or TMAO that inhibit reactivation tend to stabilize this (these) altered conformation(s).
As noted above, the pathway for folding and association presented above (Eqns 1–4, above), as formulated by Jaenicke and coworkers [15,27], postulates that the final step in the pathway is:
where T, the tetramer, is the only enzymatically active species. In light of the results presented in Fig. 8 and Table 2, perhaps step four of the pathway should be revised, and an additional step should be added as follows:
where T* represents the non-native tetramer and T represents the native enzyme.
Compelling evidence for the existence of tetrameric species having altered conformations during the early stages of the reassociation of bovine and porcine LDH polypeptides was presented by King and Weber . The enzyme dissociates at high hydrostatic pressure, generating enzymatically inactive subunits having diminished affinity for one another; on decompression the tetramer forms rapidly, but due to slow reversal of the conformational drift that occurs upon reassociation, recovery of activity occurs on a much slower time scale. The results presented in Fig. 8 and Table 2 are consistent with such a model.
The effects of TMAO and proline on the rate and extent of reactivation following denaturation in GdnHCl were qualitatively similar to those observed with acid-dissociated enzyme. Reactivation following unfolding in the chaotropic agent, however, was far more sensitive to the osmolytes (compare Fig. 3 with Figs 4B and 6). For example, inhibition of the extent of reactivation of the GdnHCl-treated enzyme by 4 m proline was approximately 75%, but only 15% for the enzyme inactivated at low pH. TMAO was the more potent inhibitor; concentrations of TMAO up to 1 m were virtually without effect on the extent of reactivation of the acid-dissociated enzyme (Fig. 6), but inhibition of the extent of reactivation following unfolding in GdnHCl by 500 mm TMAO was very significant and was almost complete in 1 m TMAO; equivalent inhibition by proline required 5 m osmolyte (Fig. 3).
As with acid-dissociated protein, it seems likely that inhibition of reactivation following unfolding in GdnHCl by these osmolytes arises from stabilization of non-native intermediates in the reactivation pathway. Due to extensive unfolding by the chaotropic agent, the potential for interaction with sidechains that are not exposed to solvent in the sodium sulfate stabilized subunits of the acid-dissociated protein, as well as those that lie at the interfaces of the subunits, may contribute to the greater osmolyte sensitivity of reactivation from GdnHCl. Interaction among one or more of these molecular species and the postulated multimeric proline species may also contribute to the inhibitory effects of this osmolyte.
Viscosity undoubtedly also plays a role in inhibition by proline of reactivation of LDH following denaturation in GdnHCl. However, the greater complexity of the reactivation pathway precludes identification of the specific molecular transitions that may be sufficiently large to be affected by the hydrodynamic properties of the solute; some of these are likely to be more viscosity-sensitive than others. Thus, it is perhaps not surprising that with enzyme unfolded by the chaotropic agent, inhibition becomes significant at somewhat lower concentrations of proline than for acid-dissociated enzyme (compare Figs 2 and 4B). The unfolded molecular species produced in the chaotropic agent is perhaps a better model for the nascent polypeptide during biosynthesis than the acid-dissociated enzyme.
The observations reported here are somewhat similar to results reported by Singer and Lindquist that established a direct link between accumulation of trehalose and thermotolerance in yeast . It was further shown that trehalose both stabilized yeast proteins and attenuated the aggregation of denatured yeast proteins in vivo. Furthermore, in vitro studies showed that trehalose (0.5 m, corresponding to physiological levels attained in yeast cytoplasm) stabilized an inactive, partially folded conformation of denatured luciferase [7,8]. In the current investigation we found that 0.5 m trehalose slightly inhibited the initial rate of reactivation of BHLDH following denaturation in GdnHCl (data not shown).
Several relatively recent reports describe effects of proline on refolding of monomeric proteins following denaturation in GdnHCl. Aggregation of lysozyme was largely prevented, and significant regeneration of biological activity was observed, when renaturation was performed in the presence of 2 m proline . Similar results were obtained with carbonic anhydrase . In both instances, it was postulated that a major factor in the prevention of aggregation of folding intermediates was interaction of exposed residues with the putative supramolecular assembly of proline molecules (see above).
A more compact, native-like conformation of reduced and carboxyamidated ribonuclease A was also strongly favored in the presence of several osmolytes, including proline and TMAO; the latter was the most effective  and stabilization of the folded conformation was mediated by the osmophobic effect (see above).
There are a few reports on the stabilizing effects of proline on the structure and/or function of LDH (e.g. ); proline stabilizes the somewhat unstable isoform from rabbit muscle against several stress conditions, including repeated freezing and thawing, as well as thermal and GdnHCl-induced inactivation. Although not excluding involvement of preferential hydration , it was suggested that there are direct interactions between the hydrophobic portion of the pyrrolidine ring of postulated proline multimers and exposed nonpolar patches on the surface of LDH subunits.
With regard to the current study, perhaps the most relevant reports of the effects of proline and TMAO on the structure and/or function of lactate dehydrogenase are based on investigations performed by Bolen and coworkers [3,44,45]. A study of the effect of proline on the catalytic activity of LDH from rabbit muscle provided evidence for interaction between proline and native enzyme ; although neither Km nor Vm were altered by proline in the concentration range up to 2 m, when enzymatic activity was determined at 3 and 4 m proline, activity was significantly reduced. It should be noted that the diminished enzymatic activity in solutions containing proline was most pronounced in the concentration range where the viscosity of the solvent is sharply increased. We observed a similar effect of high proline concentration (> 1 m) on the kinetic properties of BHLDH (data not shown).
Trimethylamine oxide (= 600 mm) exhibited modest effects on the kinetic parameters of LDH from rabbit muscle that were consistent with its role in counteracting the effects of urea , and offered powerful protection against urea-induced dissociation and inactivation . There have been no reports of similar investigations of the major isoform of LDH from cardiac muscle.
It is to be expected that there are significant differences between native LDH and the unfolded (GdnHCl), or acid-dissociated, protein in the nature and number of residues that are exposed to solute. However, in light of the TMAO-induced perturbations of protein folding observed in the current study, it is perhaps significant that in short-term experiments, TMAO generally stabilized native LDH from rabbit muscle, but long-term exposure to osmolyte alone decreased the half time of inactivation by a factor of two .
While this manuscript was in preparation, a report appeared showing that high concentrations of several osmolytes, including proline (> 1 m), inhibited the reactivation of creatine kinase following denaturation in GdnHCl . Thus, the results with this oligomer (a dimer) are qualitatively similar to our observations. However, it was also found that lower concentrations of proline (< 1 m), enhanced the rate and extent of reactivation of creatine kinase. In the limited instances in which we have examined the effect of low proline concentrations (< 1 m) on the reactivation of GdnHCl- denatured LDH, the effect was insignificant. The observations of Ou and coworkers  are somewhat reminiscent of our observations showing that the initial rate of reactivation of acid-dissociated PHLDH (but not BHLDH) was enhanced by 1 m, but inhibited by 2 m proline (Fig. 5).
We are indebted to Dr Carl Frieden for helpful discussions of viscosity-dependent processes and Dr Tom Smith for analysis (by the program MS) of the surface buried in each subunit upon formation of the tetramer. We are grateful to Elsevier Science for permission to include in Fig. 2 the values for the viscosity of proline solutions that were taken from Fig. 2 of Schobert B and Tschesche H. (1978) Unusual solution properties of proline and its interaction with proteins. Biochim. Biophys. Acta,541, 270–277.