Degradation of protein disulphide bonds in dilute alkali.

The degradation of S--S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S--S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S--S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S--S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S--S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S--S bonds of some other proteins. The initial product of S--S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

Polymorphic phase behavior of unsaturated lysophosphatidylethanolamines: a 31P NMR and X-ray diffraction study

The polymorphic phase behavior of aqueous dispersions of 1-oleoyl-, 1-linoleoyl-, and 1-linolenoyl-sn-3-glycerophosphoethanolamine (1-C18:1c-PE, 1-C18:2c-PE, and 1-C18:3c-PE, respectively) has been investigated by 31P NMR, small-angle and wide-angle X-ray diffraction, and freeze-fracture techniques in response to changes in temperature and pH. Between -20 and 0 degrees C at pH 7, NMR and X-ray data indicate that 1-C18:1c-PE adopts a lamellar phase. Above 20 degrees C, the X-ray diffraction from 1-C18:1c-PE reveals no long-range lattice order, whereas the NMR data indicate lamellar structure to 90 degrees C. Freeze-fracture electron microscopy shows that 1-C18:1c-PE at pH 8.2 forms closed multilamellar vesicles upon dispersion and also that large unilamellar vesicles produced by extrusion techniques (LUVETs) can be made from 1-C18:1c-PE at pH 7. Such LUVETs can trap [3H]inulin and support a K+ diffusion potential for up to 4 h. At pH 8.5 and above, 1-C18:1c-PE forms optically clear, fluid dispersions with NMR and X-ray characteristics consistent with a micellar (noninverted) phase structure. Attempts to prepare LUVETs from 1-C18:1c-PE at pH 9 result in structures that can neither trap [3H]inulin nor support a membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic basis for the stabilities of three CutA1s from Pyrococcus horikoshii,Thermus thermophilus, and Oryza sativa, with unusually high denaturation temperatures

In order to elucidate the stabilization mechanism of CutA1 from Pyrococcus horikoshii (PhCutA1) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutA1 proteins from Thermus thermophilus (TtCutA1) and Oryza sativa (OsCutA1) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutA1, TtCutA1 and OsCutA1 were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutA1 and OsCutA1 showed that the denaturation temperatures were remarkably high, 112.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutA1 showed higher stability to both heat and denaturant than PhCutA1. Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutA1 could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutA1 basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutA1 is superior to those of the other two CutA1s over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutA1 contribute to the unusually high stability at neutral pH.     

空肠弯曲菌肠毒素理化特性的研究

经ＳＤＳＰＡＧＥ 分析发现,空肠弯曲菌细胞紧张性肠毒素（Ｃｙｔｏｔｏｎｉｃ ｅｎｔｅｒｏｔｏｘｉｎ ,ＣＥ） 的硫酸胺盐析粗提物除有一条６８ｋＤ 的带外,还有一些未分开的小分子物质,而经神经节苷脂ＧＭ１ 亲和层析后仅有６８ＫＤ 的一条带,即表明６８ｋＤ 的蛋白质为ＣＥ 的主要成分。ＣＥ 不耐热、ｐＨ 依赖和对胰酶有抗性。５６ ℃和６０ ℃加热３０ｍｉｎ 、１００ ℃加热１５ｍｉｎ 即可完全失活。其活性在ｐＨ６－０ 时最高,在ｐＨ３－０ 和９－０ 时均可使其完全丧失活性。在４ ℃保存超过３ｄ 后,其活性迅速降低。抗ＬＴ 血清能完全抑制ＣＥ 的活性。     

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.     

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.     

The effect of mild alkali and alkaline borohydride on the carbohydrate and peptide moieties of fetuin

In the light of recent reports, based on radioactive labelling studies, that substantial amounts of N-linked oligosaccharides are released from protein under the mild-alkaline borohydride degradation conditions that are usually used to release O-linked oligosaccharides, we have investigated by chemical methods the effects of alkali alone and alkaline borohydride on the carbohydrate and peptide moieties of fetuin. The chromatographic profiles on Sephadex G50 columns, of the hexose- and ninhydrin-positive components of the native and Pronase-treated glycoprotein have been compared with those obtained after treatment with mild alkali alone (0.05 M-NaOH, 50 degrees C, 16 h) or mild-alkaline borohydride (0.05 M-NaOH containing 1 M-NaBH4, 50 degrees C, 16 h). Composition and methylation analyses have been performed on carbohydrate-containing peaks and the following conclusions were drawn: mild alkali treatment alone liberated a minor hexose- and ninhydrin-positive component and mild-alkaline borohydride treatment gave a major hexose-containing peak: both of these co-chromatographed on a Sephadex G50 column with Pronase glycopeptides. The polypeptide backbone was totally broken down by the alkaline borohydride treatment. The presence of released N-linked chains after alkaline borohydride treatment was confirmed. However, from the carbohydrate composition it was calculated that no more than 10-20% of the N-linked chains were released from protein. The results of methylation analysis have raised the possibility that this release is in part due to cleavage of the chitobiosyl core.     

Unfolding rates of globular proteins determined by kinetics of proteolysis

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.     

Inhibition of Listeria monocytogenes by fatty acids and monoglycerides.

Fatty acids and monoglycerides were evaluated in brain heart infusion broth and in milk for antimicrobial activity against the Scott A strain of Listeria monocytogenes. C12:0, C18:3, and glyceryl monolaurate (monolaurin) had the strongest activity in brain heart infusion broth and were bactericidal at 10 to 20 micrograms/ml, whereas potassium (K)-conjugated linoleic acids and C18:2 were bactericidal at 50 to 200 micrograms/ml. C14:0, C16:0, C18:0, C18:1, glyceryl monomyristate, and glyceryl monopalmitate were not inhibitory at 200 micrograms/ml. The bactericidal activity in brain heart infusion broth was higher at pH 5 than at pH 6. In whole milk and skim milk, K-conjugated linoleic acid was bacteriostatic and prolonged the lag phase especially at 4 degrees C. Monolaurin inactivated L. monocytogenes in skim milk at 4 degrees C, but was less inhibitory at 23 degrees C. Monolaurin did not inhibit L. monocytogenes in whole milk because of the higher fat content. Other fatty acids tested were not effective in whole or skim milk. Our results suggest that K-conjugated linoleic acids or monolaurin could be used as an inhibitory agent against L. monocytogenes in dairy foods.     

Inhibition of Listeria monocytogenes by fatty acids and monoglycerides.

Fatty acids and monoglycerides were evaluated in brain heart infusion broth and in milk for antimicrobial activity against the Scott A strain of Listeria monocytogenes. C12:0, C18:3, and glyceryl monolaurate (monolaurin) had the strongest activity in brain heart infusion broth and were bactericidal at 10 to 20 micrograms/ml, whereas potassium (K)-conjugated linoleic acids and C18:2 were bactericidal at 50 to 200 micrograms/ml. C14:0, C16:0, C18:0, C18:1, glyceryl monomyristate, and glyceryl monopalmitate were not inhibitory at 200 micrograms/ml. The bactericidal activity in brain heart infusion broth was higher at pH 5 than at pH 6. In whole milk and skim milk, K-conjugated linoleic acid was bacteriostatic and prolonged the lag phase especially at 4 degrees C. Monolaurin inactivated L. monocytogenes in skim milk at 4 degrees C, but was less inhibitory at 23 degrees C. Monolaurin did not inhibit L. monocytogenes in whole milk because of the higher fat content. Other fatty acids tested were not effective in whole or skim milk. Our results suggest that K-conjugated linoleic acids or monolaurin could be used as an inhibitory agent against L. monocytogenes in dairy foods.     

Non-enzymatic hinge region fragmentation of antibodies in solution

Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.     

Effects of rearing temperature on sympathoadrenal activity in young adult rats

Animals reared at 18 degrees C exhibit enhanced innervation of brown adipose tissue (BAT) and greater cold tolerance as adults, yet gain more weight when fed an enriched diet compared with rats reared at 30 degrees C. To explore this paradox, sympathoadrenal activity was examined using techniques of [(3)H]norepinephrine ([(3)H]NE) turnover and urinary catecholamine excretion in male and female rats reared until 2 mo of age at 18 or 30 degrees C. Gene expression in BAT was also analyzed for several sympathetically related proteins. Although [(3)H]NE turnover in heart did not differ between groups, [(3)H]NE turnover in BAT was consistently elevated in the 18 degrees C-reared animals, even 2 mo after removal from the cool environment. Gene expression for uncoupling proteins 1 and 3, GLUT-4, leptin, and the alpha(1A)-adrenergic receptor was more abundant in BAT and the increase in epinephrine excretion with fasting suppressed in 18 degrees C-reared animals. These studies demonstrate that obesity consequent to exposure to 18 degrees C in early life occurs despite tonic elevation of sympathetic input to BAT. Diminished adrenal epinephrine responsiveness to fasting may play a contributory role.     

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.     

Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Ionization of tyrosine and lysine residues in native and modified horse cytochrome c.

1H-n.m.r. and 13C-n.m.r. spectroscopy of horse cytochrome c and 1H-n.m.r. spectroscopy of the lysine-modified proteins N epsilon-acetimidyl-, N epsilon-amidino-, N epsilon-trifluoroacetyl- and N epsilon-maleyl-cytochrome c have shown that, although the lysine modifications do not greatly perturb the protein structure at pH7 and 27 degrees C, at higher temperature or at alkaline pH some parts of the structure are markedly perturbed. At pH7 and 27 degrees C the region of the protein about Ile-57 is affected in all the modified proteins, though not all to the same degree. N epsilon-Maleylation most seriously affects the protein structure, and the fully maleylated protein is readily unfolded. At 27 degrees C all four of the tyrosine residues of native horse cytochrome c have pKa values above 11, but in N epsilon-acetimidyl-cytochrome c the pKa of one tyrosine residue is 10.2.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)