Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

31P NMR study of changes in the intracellular pH of Escherichia coli after freezing-thawing

Influence of two types of freezing, at -196 and -50 degrees C with following thawing of Escherichia coli cells at 37 degrees C on the value of intracellular pH has been studied by means of 31P NMR spectroscopy. All the cycles of freeze-thawing have been shown to result in acidification of intracellular medium on 1.0 unit pH apart from the freezing type. The extracellular medium (pHex) was acidified too, but the degree of pHex changes after freeze-thawing depended on the freezing depth.     

The K+-H+ exchanger, nigericin, modulates taste cell pH and chorda tympani taste nerve responses to acidic stimuli

The relationship between acidic pH, taste cell pH(i), and chorda tympani (CT) nerve responses was investigated before and after incorporating the K(+)-H(+) exchanger, nigericin, in the apical membrane of taste cells. CT responses were recorded in anesthetized rats in vivo, and changes in pH(i) were monitored in polarized fungiform taste cells in vitro. Under control conditions, stimulating the tongue with 0.15 M potassium phosphate (KP) or 0.15 M sodium phosphate (NaP) buffers of pHs between 8.0 and 4.6, KP or NaP buffers did not elicit a CT response. Post-nigericin (500 × 10(-6) M), KP buffers, but not NaP buffers, induced CT responses at pHs ≤ 6.6. The effect of nigericin was reversed by the topical lingual application of carbonyl cyanide 3-chloro-phenylhydrazone, a protonophore. Post-nigericin (150 × 10(-6) M), KP buffers induced a greater decrease in taste cell pH(i) relative to NaP buffers and to NaP and KP buffers under control conditions. A decrease in pH(i) to about 6.9 induced by KP buffers was sufficient to elicit a CT response. The results suggest that facilitating apical H(+) entry via nigericin decreases taste cell pH(i) and demonstrates directly a strong correlation between pH(i) and the magnitude of the CT response.     

Effects of freezing and thawing on glycerol mutants of Escherichia coli

The effects of freezing and thawing on the viability of three glycerol mutants of Escherichia coli were determined when glycerol was absent or present in either the intracellular, extracellular, or both intra- and extracellular milieux.The recovery of nonglycerolated cells was related to the combination of freezing and thawing rates. Cell survival was significantly increased when subjected to the same rates of freezing and thawing.The ability of glycerol to protect against irreversible freeze-thawing injury was related to its cellular localization. Survival was markedly enhanced by extracellular glycerol and further increased by the presence of intracellular glycerol. However, intracellular glycerol alone failed to increase cell recovery. The rate of recovery, in respect to extracellular glycerol, was dependent upon both the rate of freezing and the combination of freezing and thawing rates.     

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.     

Characterization of high-order diphtheria toxin oligomers

In 3D domain swapping, a domain of a protein breaks its noncovalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a strongly bound dimer or higher order oligomer. For some proteins, including diphtheria toxin, 3D domain swapping may affect protein function. To explore the molecular basis of 3D domain swapping in a well-characterized protein system, domain-swapped oligomers of diphtheria toxin were produced by freezing and thawing under a variety conditions, including in various salts and buffers, and at various temperatures. Reaction yields were followed by high-performance size-exclusion chromatography. The traditional low pH pulse produced by freeze-thawing in mixed sodium phosphate buffer induces the oligomerization of DT, but the addition of alkali chloride salts was found to increase the yield in the order of Li(+) > Na(+) > K(+). Unexpectedly, oligomers also formed when DT was frozen and thawed in the presence of 1 M LiCl alone. Slower freezing and thawing of the mixture led to the production of more and larger oligomers. DT oligomers were also produced by exposure to acidic buffers, and were found by electron microscopy to adopt both linear and cyclized forms in a wide distribution of sizes. Upon the basis of these results, the model for the production of DT oligomers by freezing and thawing was expanded to include a salt-mediated pathway. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions.     

Functional changes in mitochondrial properties as a result of their membrane cryodestruction. I. Influence of freezing and thawing on succinate-ferricyanide reductase of intact liver mitochondria

The interaction of the succinate dehydrogenase complex of rat liver mitochondria with an artificial electron acceptor (K3Fe(CN)6), impermeable to the mitochondrial membrane as an index of a cryoinjury is investigated. It is shown that the freeze-thawing stimulates succinate-ferricyanide reductase (SFCR) activity of intact mitochondria. The increase of the freezing and thawing rates leads to a decrease in the released SFCR activity. The released SFCR activity after low-temperature treatment is a consequence of a nonspecific change in membrane ferricyanide permeability. The released SFCR activity decreases as the freezing and thawing rates increase.     

Molecular properties of the enzymic phytohemagglutinin of mung bean

Mung bean seeds possess a tetrameric galactose-binding protein that displays two types of activities: (a) a hemagglutinin activity, and (b) an alpha-galactosidase activity. This protein can be reversibly converted by pH changes from a tetrameric form, which possesses both enzymic and hemagglutinin activities, to a monomeric form which possesses enzymic activity only. This observation suggests that the enzymic phytohemagglutinin is an aggregated form of a monomeric alpha-galactosidase. The tetrameric alpha-galactosidase has a pH optimum of about pH 7.0, while the monomeric form displays a pH optimum of 5.6. Circular dichroism difference spectra and inhibition studies suggest that aggregation induces conformational changes in the subunits sufficient to alter their enzymatic properties. The possibility of in vivo changes in subunit equilibria, when combined with the accompanying alterations in activity, provides a new concept worthy of consideration with respect to the physiological role of phytohemagglutinins.     

α-Crystallin protects human arginosuccinate lyase activity under freeze-thaw conditions

Argininosuccinate lyase (ASL) catalyzes the conversion of argininosuccinate into arginine and fumarate, a key step in the biosynthesis of urea and arginine. ASL is a tetrameric enzyme but it dissociates into inactive dimers under low temperature conditions. This study investigates the inactivation process under low temperature conditions. Inactivation was caused by dissociation of tetrameric ASL into dimers, with increased exposure of hydrophobic areas without disturbance of the secondary structure or the microenvironment surrounding the key tryptophan residues. Most activity was retained when temperatures were changed at a rate of >1 °C/min, whilst freezing or thawing more slowly resulted in greater loss of activity. Inactivation was reduced by inclusion of α-crystallin, a structural protein found in ocular lenses and a member of the small heat-shock protein family, by stabilization of the ASL quaternary structure. In addition, α-crystallin was able to restore the function of ASL that had been inactivated by slow freezing and thawing. The effect of α-crystallin was similar to that of bovine serum albumin, suggesting that both proteins exerted their effects by hydrophobic interactions. α-Crystallin therefore acts as a cryo-preservative that protects ASL activity during freezing and thawing.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.     

Response of microbial communities of Lake Baikal to extreme temperatures

The survival rate, metabolic activity, and ability for growth of microbial communities of Lake Baikal after exposure to extremely low temperatures (freeze-thawing) for different lengths of time have been first studied. It has been shown that short-term freezing (1-3 days) inhibits the growth and activity of microbial communities. The quantity of microorganisms increased after 7- and 15-day freezing. In the periods of maximums, the total number of microorganisms in the test samples was twice as high as in the control. It was established that after more prolonged freezing the microorganisms required more time after thawing to adapt to new conditions. In the variants with 7- and 15-day freezing, the activities of defrosted microbial communities were three or more times higher than in the control. The survival rate and activity of Baikal microorganisms after freeze-thawing confirms the fact that the Baikal microbial communities are highly resistant to this type of stress impact.     

Response of microbial communities of Lake Baikal to extreme temperatures

The survival rate, metabolic activity, and ability for growth of microbial communities of Lake Baikal have been first studied after exposure to extremely low temperatures (freeze-thawing) for different lengths of time. It has been shown that short-term freezing (1–3 days) inhibits the growth and activity of microbial communities. The quantity of microorganisms increased after 7-and 15-day freezing. In the periods of maximums, the total number of microorganisms in the test samples was twice as high as in the control. It was established that after more prolonged freezing the microorganisms required more time after thawing to adapt to new conditions. In the variants with 7-and 15-day freezing, the activities of defrosted microbial communities were three or more times higher than in the control. The survival rate and activity of Baikal microorganisms after freeze-thawing confirms the fact that the Baikal microbial communities are highly resistant to this type of stress impact.     

Investigation of the role of lysine in the subunit contact regions of rabbit muscle aldolase.

Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with O-methylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 m MgCl₂ with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 m MgCl₂ nitrotroponlylation modifies another 8–12% of the lysine residues. Removal of MgCl₂ by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl₂ concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase.     

Protein-bound SH groups in frozen-thawed egg cells of the sea urchin

Unfertilized egg cells of the sea urchin St. intermedius could survive slow freezing to ?15 °C for a short period of time, but at the same freezing temperature extracellular freezing became fatal within a few hours. Such freezing injury resulted in “black” or “white” cytolysis in frozen-thawed cells. “Black” cytolysis took place in the process of both freezing and thawing, while “white” cytolysis occurred only on thawing. Rapid rewarming consistently produced “white” cytolysis in extracellularly frozen cells. The observed behavior of the injured cells during freeze-thawing appeared favorable for the explanation of freezing injury by the SH-SS hypothesis. Protein-bound SH groups were quantitatively determined in both whole cell and cortex with plasma membrane before and after freeze-thawing. However, no significant change in the SH value was observed between freeze-thaw cytolysed materials and unfrozen ones.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

Molecular properties of yeast glyceraldehyde-3-phosphate dehydrogenase in the presence of ATP and KCl.

The influence of ATP and KCl on the quaternary structure and the enzymatic activity of D-glyceraldehyde-3-phosphate dehydrogenase from yeast(Y-GAPDH) has been studied by ultracentrifugation, gel chromatography and standard optical tests. In 0.1 M imidazole buffer pH 7.0, at low temperature (0°C) both complete deactivation and dissociation to dimers occur in the presence of 2 mM ATP and 0.1 M 2-mercaptoethanol. In 0.067 M phosphate buffer pH 7.0, containing 2 mM ATP and 1 mM dithiothreitol, only slight deactivation paralleled by minor changes of the native quaternary structure is observed. In this same buffer, increasing temperature leads to stabilization of both the tetrameric state and the catalytic activity of the enzyme. Deactivation and dissociation in the presence of 0.15 M KCl (in 0.2 M glycine buffer 9.1 ≥ pH ≥ 8.0) is a function of pH rather than electrolyte concentration; at neutral pH the enzyme is stabilized in its native state. Contrary to earlier assumptions in the literature, ATP and KCl under the above experimental conditions do not appear to play an important role in the $\text{in vivo}$ regulation of Y-GAPDH.     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.