Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumoniae NCTR 1.

An amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from Klebsiella pneumoniae NCTR 1. The enzyme is a monomer with an apparent molecular weight of 62,000. The pH and temperature optima of the enzyme were 7.0 and 65 degrees C, respectively. The purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (DTNB)-titratable sulfhydryl (SH) groups. In the native enzyme 1.0 SH group readily reacted with DTNB with no detectable loss of activity. Titration of the next 3.0 SH groups with DTNB resulted in a loss of activity of more than 70%. The remaining seven inaccessible SH groups could be titrated only in the presence of 8 M guanidine hydrochloride. Titration of SH groups was strongly inhibited by carboxymethylation and KMnO4, suggesting the presence of SH groups at the active site(s). Inductively coupled plasma-atomic emission spectrometry analysis indicated that the native amidase contains 0.33 mol of cobalt and 0.33 mol of iron per mol of the native enzyme. Polyclonal antiserum against K. pneumoniae amidase was raised in rabbits, and immunochemical comparisons were made with amidases from Rhodococcus sp., Mycobacterium smegmatis, Pseudomonas chlororaphis B23, and Methylophilus methylotrophus. The antiserum immunoprecipitated and immunoreacted with the amidases of K. pneumoniae and P. chlororaphis B23. The antiserum failed to immunoreact or immunoprecipitate with other amidases.     

THE SULFHYDRYL GROUPS OF EGG ALBUMIN

1. 1 cc. of 0.001 M ferricyanide, tetrathionate, or p-chloromercuribenzoate is required to abolish the SH groups of 10 mg. of denatured egg albumin in guanidine hydrochloride or Duponol PC solution. Both the nitroprusside test and the ferricyanide reduction test are used to show that the SH groups have been abolished. 2. 1 cc. of 0.001 M ferrocyanide is formed when ferricyanide is added to 10 mg. of denatured egg albumin in neutral guanidine hydrochloride or urea solution. The amount of ferricyanide reduced to ferrocyanide by the SH groups of the denatured egg albumin is, within wide limits, independent of the ferricyanide concentration. 3. Ferricyanide and p-chloromercuribenzoate react more rapidly than tetrathionate with the SH groups of denatured egg albumin in both guanidine hydrochloride solution and in Duponol PC solution. 4. Cyanide inhibits the oxidation of the SH groups of denatured egg albumin by ferricyanide. 5. Some samples of guanidine hydrochloride contain impurities which bring about the abolition of SH groups of denatured egg albumin and so interfere with the SH titration and the nitroprusside test. This interference can be diminished by using especially purified guanidine hydrochloride, adding the titrating agent before the protein has been allowed to stand in guanidine hydrochloride solution, and carrying out the nitroprusside test in the presence of a small amount of cyanide. 6. The SH groups of egg albumin can be abolished by reaction of the native form of the protein with iodine. It is possible to oxidize all the SH groups with iodine without oxidizing many of the SH groups beyond the S-S stage and without converting many tyrosine groups into di-iodotyrosine groups. 7. p-chloromercuribenzoate combines with native egg albumin either not at all or much more loosely than it combines with the SH groups of denatured egg albumin or of cysteine. 8. The compound of mercuribenzoate and SH, like the compound of aldehyde and SH and like the SH in native egg albumin, does not give a nitroprusside test or reduce ferricyanide but does reduce iodine.     

Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.     

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation

Highly pure lysosomes were isolated from buffalo(Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-Β-glucosamindase and cathepsin D in the lysosomal preparation were 26–45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3-1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Denaturation of subtilisin BPN' and its derivatives in aqueous guanidine hydrochloride solutions.

The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible.     

Conformational study of calf brain tubulin

The conformation of calf brain tubulin has been monitored by circular dichroism, optical rotatory dispersion, and spectrophotometric titration as a function of pH, temperature, ligand concentrations, and denaturants. At pH 7, calf brain tubulin maintains its structural integrity between 5 and 37 °C as determined by circular dichroism. Furthermore, the presence of MgCl ₂ up to 1.6 × 10^?2m does not induce any observable changes in the circular dichroism spectra, nor does 10^?4m CaCl₂. With increasing pH, the spectral data can best be described as a gradual loosening of the secondary structure between pH 7 and 9. Both spectral and titrimetric data suggest a major unfolding of tubulin between pH 9 and 10. The apparent pK of tyrosine shifts from 10.85 to 9.98 upon transferring from buffer to 6 m guanidine hydrochloride, indicating that at least 14 of the 15 tyrosine groups are not fully accessible to protons in the native protein. The single disulfide bridge in calf brain tubulin helps to maintain a domain which is highly resistant to unfolding by denaturants.     

Monkey glutathione S-aryltransferases. II. Properties of the major enzyme purified from the liver.

1. The molecular and enzymatic properties of the major component (Fraction IV) of glutathione S-aryltransferases [EC 2.5.1.13] purified from the liver of monkey (mainly rhesus monkey) have been investigated. The enzyme had a molecular weight of about 48,000 and was composed of two subunits of apparently identical molecular weight (ca. 24,000) bound to each other non-covalently. Each subunit contained one SH group. The amino acid composition showed characteristic high contents of leucine and glutamic acid residues. No amino-terminal residue was detected by the dansyl method. 2. The enzyme showed a rather broad optimum pH range from 7.5 to 9 with 1,2-dichloro-4-nitrobenzene as a substrate. It was moderately stable below 40 degrees C at pH 7.5. However, it showed an anomalous instability at pH around 4.2. It was reversibly denatured at least partially by urea or guanidine hydrochloride and irreversibly denatured by sodium dodecyl-sulfate. It was significantly inhibited by Zn2+, Cd2+, and Hg2+, and also by benzene hexachloride. It was extensively inactivated by reaction with phenylglyoxal or 2,4,6-trinitrobenzene sulfonate, whereas several SH reagents were without marked effect on the activity under the reaction conditions employed.     

Two structurally different types of rabbit light chains

Disulphide bonds of rabbit γ-G-globulin and the antibody of the γ-G-globulin type against the 2,4-dinitrophenyl group were split both by the oxidative sulphitolysis at pH 8.6 and by the reduction with 2-mercaptoethanol followed by carboxymethylation. The fractionation was carried out in 0.05 m formic acid containing 6m urea, in 1m propionic acid or in 6m guanidine hydrochloride. Both heavy (H) and light) (L) chains are released from the I+J fraction preceding on an elution diagram H chains when rechromatographed in a stronger desaggregation medium. A small amount of the L chains is also released on rechromatography of the H chains (isolated from 1m propionic acid) in 6m guanidine hydrochloride. The separation of the degraded γ-G-globulin in 0.05m formic acid containing 6m urea or in 6m guanidine hydrochloride showed a separation of the L chains to two fractions differing by electrophoretic properties, peptide maps and N-terminal amino acids. However, these chains exhibit a similar molecular weight, immunoelectrophoretic behaviour and similar properties on reactivation of the antibody H chain.     

Effects of N-tricyanovinylamines on oxidative phosphorylation and level of SH-groups in rat liver mitochondria.

The effects of N-substituted tricyanovinylamines on oxidative phosphorylation as well as on glutathione and total SH group concentrations in rat liver mitochondria was studied. The N-TCVA derivatives studied (N-cyclohexyl; N-isobutyl; N-benzyl; N-phenyl; N-4-Br-phenyl; N-3-nitrophenyl) had an uncoupling effection on the oxidative phosphorylation. They stimulated the respiration of mitochondria and influenced their membrane potential. In their property as SH agents, the N-TCVA derivatives reduced the level of TSH groups of the mitochondria present in concentrations of 2 mumol/mg protein. The activity of succinate dehydrogenase was decreased by N-TCVA by 13%. N-TCVA derivatives changed the redox state of glutathione in mitochondria. This effect was observed at the concentration 0.3 mumol/mg protein. The results obtained in the present study support the view that the glutathione status is more sensitive than the total level of SH groups to incubation of mitochondria with SH agents such as N-TCVA derivatives.     

Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles.

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

Partial specific volumes and interactions with solvent components of α-globulin fromSesamum indicum L. in urea and guanidine hydrochloride

The interaction of α-globulin with urea/guanidine hydrochloride was investigated by determining the apparent partial specific volumes of the protein in these solvents. The apparent partial specific volumes were determined both under isomolal and isopotential conditions. The preferential interaction parameter with solvent components calculated were 0.08 and 0.1 g of urea and guanidine hydrochloride respectively per g protein. In both the cases the interaction was not preferential with water. The total binding of denaturant to α-globulin was calculated both for urea and guanidine hydrochloride and the correlation between experimentally determined number of mol of denaturant bound per mol of protein and the total number of peptide bonds and aromatic amino acids were found to be in excellent agreement with each other. The changes in volume upon transferring α-globulin from a salt solution to 8 M urea and 6 M guanidine hydrochloride were also calculated. This work was done at the Biochemistry Department, Brandeis University, Waltham, Massachusetts 02254, USA.     

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.0 to 8.0 causes a reduction in the apparent molecular weight of the enzyme at high KCl concentration, which can be interpreted as due to a deprotonation-induced isomerization process. Removal of Zn2+ from AMP deaminase has effect similar to alkalinization in modifying the sedimentation properties of the enzyme. In the native enzyme at high K+ concentration about 7, 9 and 12 SH groups can be titrated with Nbs2, approximately 1, 2 and 4 SH groups reacting as fast sets, at pH 6.0, 7.0 and 8.0, respectively. Substitution of the 12 SH groups reactive with Nbs2 at pH 8.0 has no effect on the pH-dependent allosteric behaviour of the enzyme. Removal of K+ causes considerable changes in the reactivity of AMP deaminase towards Nbs2, unmasking a class of additional SH groups, so that the total number of titratable SH groups approaches that of 30 determined in denaturing conditions. In the enzyme previously treated with N-ethylmaleimide to alkylate the fast reacting class of SH groups, the class of additional SH groups are substituted by Nbs2 at basic pH, but not at acidic pH, with a concomitant reduction of the enzyme activity.     

Effects of an interchain disulfide bond on tropomyosin structure: intrinsic fluorescence and circular dichroism studies

An interchain disulfide crosslink was introduced into rabbit skeletal tropomyosin (TM) at Cys190 by two different methods under non-denaturing conditions. The effects of the crosslink on the structure of tropomyosin were investigated by fluorescence and circular dichroism methods as a function of temperature and guanidine · hydrochloride concentration. Four different preparations were studied: Nbs₂-TM, red-TM crosslinked with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate); O₂-TM, TM whose SH groups were air-oxidized; red-TM, TM reduced with dithiothreitol; IA-TM, red-TM whose SH groups were blocked with iodoacetamide. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies indicated that SS crosslinks were quantitatively introduced between the subunits of TM for Nbs₂-TM and O₂-TM. In the completely folded state (below 25 °C or in the absence of denaturant) and in the unfolded state (above 65 °C or greater than 4 m-guanidine · hydrochloride) all of the samples had the same Tyr fluorescence quantum yield, accessibility to acrylamide fluorescence quenching, fluorescence polarization and mean residue rotation at 222 nm. Thermal and denaturant-induced unfolding profiles at pH 7.5 were obtained for each sample with measurements of these parameters. The main transition at about 45 °C or 2 m-guanidine · hydrochloride was shifted about +7 deg. C and 0.8 m in guanidine · hydrochloride, respectively, for the crosslinked samples as compared to the uncrosslinked samples. In addition, a destabilizing pretransition was observed in the 30 to 45 °C region or the 0 to 2 m-guanidine · hydrochloride region only for the crosslinked samples when polarization or ellipticity was measured. Studies of the ability of Nbs₂ to crosslink red-TM as a function of guanidine · hydrochloride concentration indicated that the chains separate at Cys190 between 0 and 2 m-guanidine · hydrochloride before they dissociate. Thus, the effect of the SS crosslink at Cys190 on the conformation of TM at physiological temperatures appears to be related to the inherent instability of the molecule in this region of the sequence.     

Fluorescence emission properties of S-Layer enhanced green fluorescent fusion protein as a function of temperature, pH conditions, and guanidine hydrochloride concentration

The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

SULFHYDRYL GROUPS OF EGG ALBUMIN IN DIFFERENT DENATURING AGENTS

1. The reaction between ferricyanide and egg albumin in solutions of urea, guanidine hydrochloride, and Duponol has been investigated. 2. In neutral medium ferricyanide oxidizes all the SH groups of egg albumin that give a color reaction with nitroprusside. In neutral medium ferricyanide appears to react only with the SH groups of egg albumin. The quantity of ferrocyanide formed can accordingly be considered the equivalent of the number of SH groups in egg albumin detectable with nitroprusside. 3. In solutions of urea, guanidine hydrochloride, and Duponol sufficiently concentrated so that all the egg albumin present is denatured, the same number of SH groups are found—equivalent to a cysteine content of 0.96 per cent. 4. In denaturation of egg albumin loss of solubility (solubility not in presence of the denaturing agent, but solubility examined in water at the isoelectric point) and appearance of reactive SH groups are integral parts of the same process. As denaturation proceeds in urea, SH groups are liberated only in the egg albumin with altered solubility and in this albumin the maximum number of SH groups is liberated. In a molecule of egg albumin either all of its SH groups that give a test with nitroprusside are liberated or none of them are.     

Respiration-dependent uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitochondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATPase activity was stimulated at higher concentrations of uncouplers.

2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response.

3. The addition of succinate, NADH or ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin.

4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10^-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin.

5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity.

6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

Suppression by Exogenous Phospholipid of Cyanide-Insensitive Respiration of Submitochondrial Particles from Sweet Potato Root Tissue

Submitochondrial particles from sweet potato root tissue retainedthe respiratory characteristics of the intact mitochondria withrespect to the sensitivity to cyanide and salicylhydroxamicacid. The activities of total, cyanide-insensitive, and salicylhydroxamate-sensitiverespiration of the submitochondrial particles yielded from adefinite weight of tissue slices incubated under aerobic conditions,particularly in ethylenecontaining air, were higher than thosefrom the same weight of intact tissue. The less phospholipidthe submitochondrial particles contained relative to protein,the higher the activities of cyanide-insensitive and salicylhydroxamate-sensitiverespiration tended to be relative to total respiratory activity.When the submitochondrial particles were incubated with phospholipidliposomes, the activities of cyanide-insensitive and salicylhydroxamate-sensitive,but not cyanide-sensitive, respiration became extremely low.All phospholipids showed this effect. Such incubation of thesubmitochondrial particles with phospholipid liposomes yieldedlighter particles, indicating close association of exogenouslyadded phospholipid with the particles. Phospholipid moleculesseemed to enter the membrane of the particles. We propose thatphospholipid deficiency in the mitochondrial inner membranefacilitates operation of the cyanide-insensitive electron transportpath. (Received March 30, 1984; Accepted June 15, 1984)     

Circular dichroism and gel filtration behavior of subtilisin enzymes in concentrated solutions of guanidine hydrochloride.

The circular dichroism of diisopropylphosphorylsubtilisins Novo and Carlsberg in both the near- and farultraviolet spectral regions is unaltered by concentrations of guanidine hydrochloride as high as 4 M at neutral pH. At concentrations of guanidine hydrochloride greater than 4 M slow irreversible time-dependent changes, apparently obeying second-order kinetics, are evident in both the near- and far-ultraviolet circular dichroism of these enzymes. Gel filtration studies of inactivated subtilisin enzymes reveal the circular dichroism changes to be accompained by the appearance of aggregated protein material. The changes in circular dichroism and the production of associated subtilisin species are sensitive to protein concentration, denaturant concentrations, and pH. The circular dichroism of active subtilisins Novo and Carlsberg in guanidine hydrochloride exhibits irreversible changes similar to those observed for the inactivated subtilisins. Aggregated protein material is also formed initially in the presence of guanidine hydrochloride, but is rapidly autolyzed to low molecular weight fragments.