Denaturing Reagents Research Articles

Relationship between the ligand structure of copper and the stability of superoxide dismutase.

The ligand structure of chelated copper in recombinant human Cu(2)Zn(2)SOD (r-hSOD) and its metal-substituted SODs was examined. In r-hSOD the configuration of the copper binding site was axially symmetrical and that of metal-substituted SODs had a tendency to change towards a tetrahedral type compared with r-hSOD except for Cu(2)(VO)(2)SOD. Cu(2)(VO)(2)SOD was not tetrahedral and in this case the ligand atom of copper was inclined to convert from nitrogen towards oxygen. In Cu(2)E(2)SOD, Ag(2)Cu(2)SOD, and Cu(2)(VO)(2)SOD the specific activities were lower but in CU(2)Cu(2)SOD and Cu(2)Co(2)SOD they were nearly identical with that of r-hSOD. These results suggested the ligand structure of chelated copper was not related to the specific activity of SOD. However, these SODs were less stable than r-hSOD against heat treatment and denaturing reagent. Further they were less stable against attack by an inactivator (hydrogen peroxide) except for Cu(2)(VO)(2)SOD. In this case the decreased stability of these SODs was associated with the change of the ligand structure of copper from that of r-hSOD. These results suggested that the presence of zinc contributed highly to the stable formation of the ligand structure of copper and the enzymatic stability.

A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilic HB8: purification and general properties

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermus thermophilus HB8, and shown to be a dimeric protein of molecular weight 115,000, with a pI of 5.5. The amino acid composition of the present enzyme was similar to that reported for other bacterial counterparts, except for a high Arg/Lys ratio and a low Cys content. Divalent cations, such as Mn2+ and Mg2+, were essential for activity. The optimal pH was 7.8 at 55 degrees C. The Km values for NADP and D-isocitrate were 6.3 and 8.8 microM, respectively, with a Vmax of 77.6 mumol/min per mg at 55 degrees C. NAD was able to replace NADP with low efficiency. Backward reaction at 40 degrees C indicated that the Km value for 2-oxoglutarate was 63 microM with a Vmax of 4% that of the forward reaction at that temperature. The enzyme was highly stable against high temperature and denaturing reagents.

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.

Purification and characterization of serine protease inhibitor from white croaker Argyrosomus argentatus ordinary muscle.

A serine protease inhibitor was isolated from white croaker Argyrosomus argentatus ordinary muscle by ammonium sulfate fractionation, gel filtration on Sephadex G-100, column chromato-graphies on CM-Sephadex C-50 and DEAE-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The inhibitory activity against trypsin increased about 1, 500-fold, and the purified inhibitor was homogeneous as judged by polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was estimated to be about 100, 000 by gel filtration on Sephadex G-100, but was estimated to be about 55, 000 by SDS polyacrylamide gel electrophoresis after reduction with 2-mercaptoethanol. The inhibitor was stable over a range of pH 6.0-9.0, but unstable below pH 5.0, and completely inactivated by heating at 60°C for 30 min at pH 6.0. The isoelectric point was found to be about 4.7, and N-terminal glycine was found by dansylation. Trypsin, α-chymotrypsin and elastase were strongly inhibited with the inhibitor, but pepsin, thermolysin and papain were not inhibited. Each E-I complex of the inhibitor with the proteases, trypsin, α-chymotrypsin and elastase, were stable to the denaturing reagents such as SDS and 2-mercaptoethanol, however each complex was digested in the presence of the excess protease.

Stabilization of fumarase activity ofBrevibacterium flavum cells by immobilization with k-carrageenan

Whole cells ofBrevibacterium flavum having fumarase activity were immobilized using K-carrageenan. The stabilities of fumarase activity in the immobilized cells against external factors, including heat, pH, organic solvents, and protein denaturing reagents, were compared with those of free cells and native enzyme. The stabilities of fumarase activity in immobillized cells against external factors were highest, and those of native enzyme were lowest. In the “gel-state,” K-carrageenan-immobilized cells showed a much higher stabilization effect for external factors than “sol-state” immobilized cells.

Heat-Stable Extracellular Proteolytic Enzyme Produced by Thermus caldophilus Strain GK24, an Extremely Thermophiic Bacterium1

Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).

Measurement of medium lysyl oxidase activity in aorta smooth muscle cells. Effects of multiple medium changes and inhibition of protein synthesis.

Cultures of rabbit aortic smooth muscle (RSM) cells are a valuable model system for studying production and metabolism of connective tissue components. This report describes various assay procedures for lysyl oxidase, the enzyme responsible for deaminating lysine residues to give aldehyde cross-link precursors, in culture medium from these cells. Studies of the medium enzyme from second-passage RSM cells indicate that approximately 40% of the total enzyme activity in the flask of cells is in the medium. The medium enzyme levels are replenished quite rapidly following refeeding, and enzyme levels in the medium appear to be feedback controlled. The mechanism for this control is unknown at present. Multiple refeeding experiments in which the medium was changed every 2-4 h for up to 40 h indicate that these cells are cap]able of producing large amounts of enzyme and are capable of altering enzyme production and secretion quite rapidly in response to changes in their environment. Protein synthesis inhibitor studies with cycloheximide suggest that the major portion of the enzyme released into the medium following refeeding is newly synthesized although a pool of latent enzyme is also present. As in intact tissue, extraction of the enzyme from the cell layer requires strong denaturing reagents such as 4 M urea. These results suggest that the production of lysyl oxidase is closely regulated and is very responsive to changes in the external environment of the cells. This cell culture system appears to be an excellent one to study the production of lysyl oxidase and its role in connective tissue fibrillogenesis.

A [formula omitted] procedure for the isolation of chromatin proteins (histones and nonhistones) from wheat germ

A new procedure is described for the isolation of both histone and nonhistone chromatin proteins, based on a polyethylene glycol (PEG) dextran two-phase partition system. Chromatin is solubilized in high salt (5 M NaCl) and mixed with PEG and dextran to separate proteins (partitioned into the upper, PEG-rich phase) from nucleic acids (DNA recovered almost exclusively in the lower, dextran-rich phase). The proteins are then adsorbed onto Bio-Rex 70 by dialysis to low salt (0.05 M NaCl), followed by salt elution to recover first nonhistone proteins (<0.55 M NaCl), then histones (2 M NaCl). Cross-contamination is not detectable in either group of proteins. The procedure is rapid, gentle, and lends itself well to scale-up. The proteins are kept in pH range 7.0–8.1, and are not exposed to the denaturing reagents characteristic of most preparative procedures for chromatin proteins. Though developed specifically for the isolation of proteins from chromatin of wheat germ, the procedure should be readily applicable to other sources as well. Wheat germ (isolated wheat embryos) appears to be an excellent source of chromatin proteins; 100 g yields 225–300 mg histones and 30–45 mg nonhistone proteins, depending on technique.

Sodium trichloroacetate-induced helical conformation of poly( l-lysine)

Sodium trichloroacetate which has been reported previously to be an effective denaturation reagent for proteins was applied to poly( l-lysine) and poly( l-glutamic acid) to see its effects on a coil-to-helix transition and on the chemical reactivities of the ε-amino group of poly( l-lysine). Addition of sodium trichloroacetate to poly-( l-lysine) induced a helical conformation even at neutral pH where the ε-amino group of the polymer was protonated. On the other hand, little effect was observed on the coil-to-helix transition of poly( l-glutamic acid). The ε-amino group of poly-( l-lysine) has an anomalously high reactivity with naphthoquinone 4,6-disulfonate carrying a negative charge. Sodium trichloroacetate inhibited the reaction of the ε-amino group with this reagent, while sodium trichloroacetate enhanced slightly the reaction of the ε-amino group of poly( l-lysine) with diazonium-1-H-tetrazole carrying a positive charge.

Regulatory Factors Produced by Lymphocytes

Abstract Supernatant fluids of mitogen-activated human tonsil lymphocytes contain large amounts of a factor toxic to mouse L cells. This substance, with a m.w. of 80,000 ±5,000 daltons, is called α-lymphotoxin (α-LT), to differentiate it from another toxin elaborated by mitogen-activated human blood lymphocytes, called β-lymphotoxin (β-LT), which differs from α-LT in size (45,000 ±5,000 daltons), antigenicity, and stability. Further purification of α-LT by sequential phosphocellulose and DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) identifies a series of cytotoxins differing in ion exchange characteristics and electrophoretic mobilities. The three PAGE fractions (PAGE Ia, Ib and II), recovered in 2, 4.6, and 21% yield from the starting serum-free culture supernatant, represent purifications of 24-, 24- and 1851-fold, respectively. Each cytotoxic fraction has a ribonuclease activity. Comparison of RNase and mouse L cell cytotoxic activities of the three α-LT fractions shows that both activities for all three fractions have a similar temperature stability pattern and that both are similarly inhibited by DNA, single strand forms better than double strands, by glycerol in 5 to 20% concentration, and by protein denaturing reagents. These observations suggest, but do not prove, that mouse L cell toxicity and RNase activity are mediated by the same substance, which appears to occur in multiple or isozymic forms.

Recent studies on the chemistry of human, bovine and porcine parathyroid hormones

The amino acid sequence of the NH 2-terminal 34 residues of human parathyroid hormone (PTH) has been determined and duplicated synthetically to produce a peptide that is biologically active. In the amino acid sequences of the bovine and porcine hormones, the glutamic acid function at position 22 has been revised to glutamine. Among these initial 34 residues, human PTH differs from bovine PTH by 5 residues and from porcine PTH by 4 residues. Native human PTH and the synthetic human PTH (1–34) peptide are not rigid structures, and significant changes in conformation were observed during pH titration. In addition, at physiologic pH, native human PTH appeared to differ in structure from human PTH (1–34) in the region of the tryptophan residue (residue 23). The fluorescence spectrum of human PTH revealed a maximum at 344 nm, but the spectrum of human PTH (1–34) had a peak at 343 nm; the spectrum of human PTH (1–34) was normalized to 346 nm in 6 M guanidine hydrochloride, but there was no shift with the intact hormone. Fluorescence titration of human PTH in the alkaline region revealed no loss of tryptophanyl fluorescence in aqueous solution or in 6 M guanidine hydrochloride. The synthetic human PTH (1–34) peptide, however, showed an approximately 25 per cent loss of indole fluorescence during alkaline titration which could be normalized with denaturing reagents. These studies suggest that synthetic fragments of the native hormone may not have the same tertiary conformation as the same sequence in the intact hormone. These findings may be of major significance with regard to the biologic activity and immunologic cross reactivity of synthetic fragments and the native hormone.

Physical and Chemical Properties of the Lipase of Thermophilic Fungus Humicola lanuginose S-38

Some physical and chemical properties of the extracellular lipase from the thermophilic fungus, Humico la lanuginosa S–38, were investigated. The results were as follows: Sedimentation coefficient was 2.4 × 10−13 (cm-g/sec-dyne); diffusion coefficient was 8.8 × 10−7 (cm2/sec); and frictional coefficient was 1.22. Molecular weight was 27,500±500 and α-helix content was 18.9%. The number of amino acid residues contained in 1 mole of protein of Humicola lipase was 224. Sugar and lipid were not detected. The effect of calcium ion and denaturing reagents, such as urea, sodium dodecyl sulfate and dithiothreitol, on the thermostability of Humicola lipase was examined. It was concluded that the thermostability of Humicola lipase was not influenced by protective cofactors but was attributable to the enzyme itself. Some properties of enzyme structure which were concerned with the thermostability of Humicola lipase are also discussed.

Effects of sodium trichloroacetate on the heme bindings in hemoglobin and myoglobin derivatives

Sodium trichloroacetate (Na-TCA) which was reported previously as an effective denaturation reagent for proteins was applied at neutral pH to oxy-, deoxy-, met-, and cyanomet-derivatives of hemoglobin and myoglobin. The absorption spectra of these hemoproteins were changed by addition of Na-TCA above a certain concentration. Difference in the concentration of Na-TCA giving spectral, change of hemoproteins suggested that the structure of hemoglobin changes to a greater extent than does the structure of myoglobin when they bind ligands. The affinity of heme iron atom of metmyoglobin to OH − ion was lowered by the presence of Na-TCA (0–0.6 M ). The presence of high concentration of Na-TCA (0.7–1.0 M ) decreased the absorbance at 409 mμ, the mean residue ellipticity on CD spectrum, and the k 1 and k 4 values of peroxidase activity. These changes were ascribed to the change of the structure of the heme group.

Sodium trichloroacetate as a denaturation reagent for proteins.

Trichloroacetic acid (TCA) is known as a precipitant for proteins at acidic pH was applied at neutral pH to proteins such as α-chymotrypsin [EC 3. 4. 4. 5], chymotrypsinogen, lysozyme [EC 3. 2.1.17], ribonuclease A [EC 2. 7. 7.16] and insulin in order to examine its effects on the structures and activities of these enzymes and proteins at neutral pH and to compare them with the effects of urea, guanidine-HCl and halogen derivatives of acetate. The proteolytic activity of α-chymotrypsin was inhibited completely by addition of 1.2 M Na-TCA at pH 7.8, while the activity was fully retained in the presence of 1.5 M sodium monochloroacetate, sodium dichloroacetate, sodium trifluoroacetate, urea or guanidine-HCl at the same pH value. The activities of ribonuclease A and lysozyme near neutral pH were also lost completely with Na-TCA at 1.2M. The four tyrosine residues in the α-chymotrypsin molecule ionized with 0.6M Na-TCA at pH 12.0 while, in the presence of other reagents at 1.5 M, only two or three of them ionized at the same pH value. Some tyrosine residues in chymotrypsinogen, lysozyme and ribonuclease A, which do not ionize at pH 12.0 without reagent, ionized on addition of 1.5 M Na-TCA at the alkaline pH. These proteins when treated with Na-TCA at pH 7.0 underwent spectral shifts due to exposure of tyrosine and/or tryptophan residues from the interior of protein molecules. The helical contents of ribonuclease A and lysozyme increased on addition of Na-TCA, whereas the helical content of insulin and α-chymotrypsin decreased on the same treatment. Na-TCA was thus found to act as a denaturation reagent for proteins which is more effective at neutral pH than the other notable denaturation reagents.

Studies on the chemical modification of staphylococcal enterotoxin B. II. Carboxyl residues

The carboxyl groups in staphylococcal enterotoxin B were modified with glycine methyl ester in the presence of water soluble carbodiimide. All of the 39 carboxyl groups were quantitatively modified when the reaction was carried out in the presence of 6 M guanidine · HCl. In the absence of denaturing reagent only 26–29 residues were available for reaction. These carboxyl groups were considered as “free” carboxyl groups. Modification of free carboxyl groups in enterotoxin B has a slight effect on the antigen-antibody reaction and fluorescence properties, but has little or no effect on the emetic activity. The emetic activity was lost when 35 or more carboxyl groups were modified. Expansion of enterotoxin B molecules has been observed after modification. The role of carboxyl groups on the biological and structural function in enterotoxin B is discussed.

Thermostable Protease from Thermophilic Bacteria: II. STUDIES ON THE STABILITY OF THE PROTEASE

1. The effect of temperature on the conformation of the protease from Bacillus thermoproteolyticus was examined. 2. The thermostable enzyme is stable at room temperature against such denaturing reagents as urea, alcohol, and detergent. 3. However, these reagents accelerate the denaturation of the enzyme by heat. 4. The effect of pH on the stability of the enzyme was examined, and it is suggested that the abnormally ionizing tyrosine residues contribute to the maintenance of the enzyme structure. 5. It is deduced from the physicochemical properties of the heated enzyme that the abnormal ionization and the Cotton effect of the tyrosine residues are due primarily to the hydrogen bonding of the phenol group.

Optical rotatory dispersion and spectrophotometric studies on fetuin in solutions containing detergent and denaturing reagents.

Abstract The effects of the denaturing agents, urea and guanidine·HCl, and detergents, sodium dodecyl sulphate and hexadecyltrimethylammonium bromide (HTAB), on the optical rotatory dispersion of fetuin have been investigated. It was concluded that fetuin has a high affinity for the cationic detergent HTAB which seems to bind, at one type of binding site only, as demonstrated by both an optical rotatory and Optical density study. The same detergent also increased by the parameters λc and b o , determined from Yang-Doty plots and the Moffitt equation, respectively. The conformation-dependent Cotton trough also shows an increase in amplitude and in addition undergoes a shift to higher wavelengths upon addition of HTAB. Although these observations suggest an increase in helical structure, caution was exercised in interpreting the results in this way since optical artifacts (e.g., higher refractive indexforprotein-micelle complex) may contribute. Modification of the fetuin molecule by removal of sialic acid or cleavage of the disulfide bonds results in a reduction in helical content of the molecule but does not alter the affinity of the protein for HTAB. The effect of HTAB on the various fetuin preparations was opposed by guanidine·HCl but not by urea; this was attributed to a charge effect by the former ions. The red shifts of the Cotton curve and the absorption spectra, upon the addition of HTAB to fetuin solutions are gradual and seem to be related, and it is tempting to attribute both phenomena to possible micelle formation in which tyrosine chromophores are also included. Such protein-micellar complexes would have a higher refractive index than the bulk solvent, and this in itself would cause a red shift of the π → π * absorption band of the helix. On the basis of solvent perturbation and spectrophotometric studies, it was concluded that fetuin contains no buried tyrosine groups. A structural similarity of both bovine albumin and fetuin was proposed as a result of the appearance in both cases of a second Cotton trough in the far-ultraviolet region after cleaving the disulfide bonds, indicative in both cases of a more random conformation.

Studies on beta-parotin. VI. The effect of various chemical reagents and proteolytic enzymes upon beta-parotin.

Beta parotin was found to be more stable in acid solution than parotin, and equally or slightly unstable in alkaline solution; it was fairly stable between pH 2.0 and 10.0 below 40°C.Ca-activity of β-parotin was inactivated by nitrous acid, formaldehyde, cysteine and thioglycollic acid, but not by iodine. It was not influenced either such denaturing reagents as urea and guanidine.Ca-activity of β-parotin was destroyed after the treatment with proteases for to 24hrs. at 38°C, while L-activity was stable.

The hyperchromic effect of organic denaturing reagents on deoxyribonucleic acid.

1. The hyperchromic effects of varying concentrations of organic denaturing reagents on the ultraviolet absorption of calf-thymus DNA and of hearing-sperm DNA have been studied at room temperature and over a range of pH values. 2. N,N-Dimethylformamide and N-methylpyrrolidone cause a hyperchromic shift at neutral and alkaline pH values; carboxylic acids (formic, acetic and propionic acids) cause one at acidic pH values; and aliphatic amines (isopropylamine and ethylamine) at alkaline pH values. 3. The percentage increments of the absorbancy at 260 mμ, produced by these reagents, are as high as 49–54% which is approximately equal shift (48–49 %) obtained by heat, and is greater than that (27–31%) obtained by inorganic acid or alkali for the same preparations of DNA. 4. The denaturations effected by these reagents are partially reversible as measured by the hyperchromic shift. 5. The absorbancy-concentrations curves of native DNA show an abrupt change of absorbancy over a narrow range of concentration of reagent which is characteristic of the unique secondary structure of DNA. The curve for the slowly-cooled sample of heat-denatured DNA shows two changes of absorbancy; a linear and gradual increase at low reagent concentrations and an abrupt change at higher concentrations. The curve for the quickly cooled sample shows the linear increase over a wide range of concentration, but no abrupt change.

The hyperchromic effect of organic denaturing reagents on deoxyribonucleic acid

1. 1. The hyperchromic effects of varying concentrations of organic denaturing reagents on the ultraviolet absorption of calf-thymus DNA and of hearing-sperm DNA have been studied at room temperature and over a range of pH values. 2. 2. N, N-Dimethylformamide and N-methylpyrrolidone cause a hyperchromic shift at neutral and alkaline pH values; carboxylic acids (formic, acetic and propionic acids) cause one at acidic pH values; and aliphatic amines (isopropylamine and ethylamine) at alkaline pH values. 3. 3. The percentage increments of the absorbancy at 260 mμ, produced by these reagents, are as high as 49–54% which is approximately equal shift (48–49 %) obtained by heat, and is greater than that (27–31%) obtained by inorganic acid or alkali for the same preparations of DNA. 4. 4. The denaturations effected by these reagents are partially reversible as measured by the hyperchromic shift. 5. 5. The absorbancy-concentrations curves of native DNA show an abrupt change of absorbancy over a narrow range of concentration of reagent which is characteristic of the unique secondary structure of DNA. The curve for the slowly-cooled sample of heat-denatured DNA shows two changes of absorbancy; a linear and gradual increase at low reagent concentrations and an abrupt change at higher concentrations. The curve for the quickly cooled sample shows the linear increase over a wide range of concentration, but no abrupt change.