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At least 19 recordsLinked to original sources
PubMed1975

Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues.

A model is proposed to account for the observation that the denaturation of small proteins apparently occurs in two kinetic phases. It is suggested that only one of these phases--the fast one--is actually an unfolding process. The slow phase is assumed to arise from the cis-trans isomerism of proline residues in the denaturated protein. From model compound data, it is shown that the expected rate for isomerism is in satisfactory agreement with the rates actually observed for protein folding. It is also shown that a simple model of protein unfolding based on the isomerism concept is very successful in accounting for many known experimental characteristics of the kinetics and thermodynamic of protein denaturation. Thus, the model is able to predict that two kinetic phases will be seen in the transition region while none are seen in the base-line regions, that both the fast and slow refolding phases lead to the native protein as the product, that the fast phase becomes the only observable phase for jumps ending far in the denatured base-line region, that most or all small proteins show a limiting low-temperature activation energy of ca. 20,000 cal, and that the relaxtion time for the slow phase seen in cytochrome c denaturation is much shorter than for all other small proteins. By utilizing "double-jump" experiments, it is shown directly that the slow phase is not part of the unfolding process but that it corresponds to a transition among two or more denatured forms which have identical spectroscopic (286.5 nm) properties. Thus, the slow relaxation is "invisible" except in the transition region where it couples to the fast unfolding equilibrium. Finally, since the present model assumes that only one of the major kinetic phases seen in denaturation reactions is concerned with the denaturation process per se, it is in agreement with numerous thermodynamic studies which show consistency with the two-state model for unfolding.

Calorimetry↗
PubMed1976

The fluorescence decay of tryptophan residues in native and denatured proteins.

The fluorescence decay kinetics at different ranges of the emission spectrum is reported for 17 proteins. Out of eight proteins containing a single tryptophan residue per molecule, seven proteins display multiexponential decay kinetics, suggesting that variability in protein structure may exist for most proteins. Tryptophan residues whose fluorescence spectrum is red shifted may have lifetimes longer than 7 ns. Such long lifetimes have not been detected in any of the denatured proteins studied, indicating that in native proteins the tryptophans having a red-shifted spectrum are affected by the tertiary structure of the protein. The fluorescence decay kinetics of ten denatured proteins studied obey multiexponential decay functions. It is therefore concluded that the tryptophan residues in denatured proteins can be grouped in two classes. The first characterized by a relatively long lifetime of about 4 ns and the second has a short lifetime of about 1.5 ns. The emission spectrum of the group which is characterized by the longer lifetime is red shifted relative to the emission spectrum of the group characterized by the shorter lifetime. A comparison of the decay data with the quantum yield of the proteins raises the possibility that a subgroup of the tryptophan residues is fully quenched. It is noteworthy that despite this heterogeneity in the environment of tryptophan residues in each denatured protein, almost the same decay kinetics has been obtained for all the denatured proteins studied in spite of the vastly different primary structures. It is therefore concluded that each tryptophan residue interacts in a more-or-less random manner with other groups on the polypeptide chain, and that on the average the different tryptophan residues in denatured proteins have a similar type of environment.

Hydrogen-Ion Concentration↗
PubMed1976

Does Freund's adjuvant denature protein antigens? EPR studies of emulsified hemoglobin.

Use of complete Freund's adjuvant for production of antibodies to study protein conformation is valid only if emulsification in adjuvant does not denature protein antigens. Using electron paramagnetic resonance to observe directly the protein in situ in the opaque emulsions, we demonstrate that hemoglobin is not denatured by emulsification or storage in adjuvant for 24 hr at 4 degrees C, conditions comparable to the usual handling of antigens before immunization.

Antigens↗
PubMed1977

Effect of chaotropic salts and protein denaturants on the thermal stability of mouse fibroblast interferon.

Altering the aqueous environment, especially with agents that affect hydrogen bonds, markedly affects the stability of mouse L cell interferon. Low pH stabilizes interferon whereas high pH labilizes it; heavy water further enhances interferon thermostability at pH 2 but not at pH 9. Exposure to the protein denaturants, 4 M-guanidine hydrochloride and 6 M-urea, significantly decreases the activity of interferon at pH 2 and pH9; however, the residual interferon activity is relatively thermostable. Certain chaotropic salts protect interferon against thermal destruction, and in terms of effectiveness, their sequence is in the order SCN- greater than 1- larger than or equal to Cl- = CiO4- - Br- greater than NO3-. Interferon becomes more stable to heat as the NaSCN concentration is increased from 0-25 M to 2-0 M. Molecular sieve chromatography of interferon in the presence of 1-5 M-NaSCN at pH 7 shows a shift in its apparent mol. wt. from 25000 to 42000. Unlike most proteins, the unfolded conformation of interferon appears to be more stable to heat than the molecule with a smaller Stokes' radius.

Deuterium↗
PubMed1976

[Nitrotyrosyl]cytochrome c. Studies of the effect of iron binding, protein denaturants and oxidation-reduction potentials.

Static measurements of the reaction of ligand binding were done by conventional spectrophotometry. The ligand-binding reactions with nitrated cytochrome c were performed with imidazole, iminazole, CO and NO. The stoicheiometry was found to be 1:1, and the stability constants for the complexes formed between the nitrated cytochrome c and the ligands are: 2.58 X 10(4) M-1 (imidazole); 1.01 X 10(2) M-1 (iminazole); 3.6 X 10(4) M-1 (CO); 2.74 X 10(4) M-1 (NO). It was found that the electrometric potentials at pH 7.0 and 25degreesC of [aminotyrosyl]cytochrome c are E'o form II = 0.115 V and E'o form I = 0.260 V, where forms I and II are two species of protein co-existing in the protein solution. The isoelectric point for the oxidized form of [nitrotyrosyl]cytochrome c was 10.05, at 4degreesC.

Carbon Monoxide↗
PubMed1978

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.

Alcohol Oxidoreductases↗
PubMed1975

An electrophoretic study of reversible protein denaturation: chymotrypsinogen at high pressures.

When reversible denaturation of chymotrypsinogen is produced at elevated hydrostatic pressures, conformational relaxation can occur quite slowly, allowing electrophoretic separation of the principal states from the equilibrium mixture. In this work we report experimental concentration distribution patterns obtained at pH 2.03 at a temperature of 20.5 degrees and find them to be reasonably consistent with the behavior that is expected from a simple two-state isomerism. However, the results do not at all rule out the existence of low levels of intermediate states.

Chymotrypsinogen↗
PubMed1975

Antigenicity and cross-reactivity of denatured fibril proteins of primary, secondary, and myeloma associated amyloids.

Antisera have been produced against amyloid fibril preparations of primary, secondary, and myeloma origins. These have revealed the presence of both idiotypic and shared antigenic determinants. A high degree of cross-reactivity among the 3 types of amyloid has been found irrespective of classification. One antiserum to a secondary amyloid fibril preparation proved to be directed against A protein (the major nonimmunoglobulin constituent of secondary amyloid fibrils). This antiserum detected A protein in several of the secondary amyloids as well as in two primary amyloid fibril preparations which were degraded with alkali; These finding indicate that amyloid fibrils of various origins have similarities in composition and, therefore, may have common factors involved in fibrillogenesis.

Amyloid↗
PubMed1977

Structural changes and fluctuations of proteins. II. Analysis of the denaturation of globular proteins.

The statistical thermodynamic model of protein structure proposed in paper I is developed with special attention to the hydrophobic interaction. Calorimetric measurements of the thermal denaturation of five globular proteins, ribonuclease A, lysozyme, alpha-chymotrypsin, cytochrome c, and myoglobin, are quantitatively analyzed using the model. The thermodynamic parameters obtained by the least squares method reflect the global, average properties of proteins and are in good agreement with the expected values estimated from experimental and theoretical studies for model peptides. The average bond energy epsilon is well related to the tertiary structure of each protein. However, the difference in the parameters between different proteins is not observed for the cooperative energy ZJ and the chain entropy alpha. The individuality of a protein as far as its structural stability is concerned, is mainly reflected by the parameter gamma specifying the hydrophobic nature of a protein. The model is further applied in the analysis of several aspects of the structural stability of globular proteins. Denaturation induced by denaturants, salts, and pH are also explained by the model in a unified manner.

Calorimetry, Differential Scanning↗
PubMed1978

Renaturation of a reduced Taiwan cobra cardiotoxin.

Refolding of a denatured protein obtained by reducing cardiotoxin from the Taiwan cobra with mercaptoethanol has been carried out in aqueous and non-aqueous solutions. Oxidation of the reduced protein in 0.05 M phosphate buffer (pH 7.2) resulted in isolating an active protein which showed, as compared to native cardiotoxin, identical conformation and biological activities such as lethality, antigenicity and muscle contracture inducing activity. On the other hand, the reduced protein was undergoing incorrect SS-pairing and several inactive products were formed in a mixture of 1,2-ethanediol and 1-propanol (1 : 1; v/v).

Animals↗
PubMed1979

Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.

A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.

Animals↗
PubMed1975

Digestion of insulin derivatives with subtilisin: a kinetic study.

Native, denatured, performic acid-oxidized or S-sulfo insulin and S-sulfo or performic acid-oxidized A- and B-chains were digested with subtilisin type Carsberg. The proteolysis was followed by measuring the uptake of alkali through autotitration. The kinetic study shows the existence of 2 first-order reaction classes which differ markedly in rate constant. The number of bonds split with fast and with slow reactions has been calculated. Only one of a total of 12 cleavable bonds in native insulin is opened by fast reaction. In the denatured protein the number of bonds split by the fast reaction increases to 4 and in the oxidized and S-sulfo protein 3 bonds are cleaved, while the slow cleavable bonds number 2 and 7, respectively, The kinetic study of the proteolysis of S-sulfo A-chain and of oxidized or S-sulfo B-chain shows that two bonds are split in A-chain with the fast and slow reactions, while in B-chain only one of the six cleavable bonds is susceptible to fast attack.

Hydrogen Bonding↗
PubMed1978

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.

Amino Acids↗
PubMed1979

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.

Animals↗