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Saigo, S. (1981). Kinetic and equilibrium studies of alkaline isomerization of vertebrate cytochromes c. Biochim Biophys Acta, 669(1), 13–20.
Abstract: Equilibria and kinetics of alkaline isomerization of seven ferricytochromes c from vertebrates were studied by pH-titration and pH-jump methods in the pH region of 7-12. In the equilibrium behavior, no significant difference was detected among the cytochromes c, whereas marked differences in the kinetic behavior were observed. According to the kinetic behavior of the isomerization, the cytochromes c examined fall into three classes: Group I (horse, sheep, dog and pigeon cytochromes c), Group II (tuna and bonito cytochromes c) and Group III (rhesus monkey cytochrome c). The kinetic results are interpreted in terms of the sequential scheme: Neutral form in equilibrium with fast Transient form in equilibrium with slow Alkaline form where the neutral and alkaline forms are the species stable at neutral and alkaline pH, respectively, and the transient form is a kinetic intermediate. From comparison of the primary sequences of the seven cytochromes c and the classification of these cytochromes c, it is concluded that the amino acid substitution Phe/Tyr at the 46-th position has a major influence on the kinetic behavior. In Group II and III cytochromes c, the ionization of Tyr-46 is suggested to bring about loosening of the heme crevice and thus facilitate the ligand replacement involved in the isomerization.
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Sukhomlinov, B. F., Korobov, V. N., Gonchar, M. V., Datsiuk, L. A., & Korzhev, V. A. (1987). [Comparative analysis of the peroxidase activity of myoglobins in mammals]. Zh Evol Biokhim Fiziol, 23(1), 37–41.
Abstract: Studies have been made on the peroxidase activity of metmyoglobins in animals from various ecological groups--the horse Equus caballus, cattle Bos taurus, beaver Castor fiber, otter Lutra lutra, mink Mustela vison and dog Canis familiaris. It was found that the level of this activity in diving animals depends on the duration of their diving, whereas in terrestrial species--on the strength of muscular contraction.
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Hirota, S., Suzuki, M., & Watanabe, Y. (2004). Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c. Biochem Biophys Res Commun, 314(2), 452–458.
Abstract: Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.
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Abbruzzetti, S., Viappiani, C., Small, J. R., Libertini, L. J., & Small, E. W. (2001). Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique. J Am Chem Soc, 123(27), 6649–6653.
Abstract: We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H(+)], which can be more than 100 microM, is complete within a few nanoseconds. The increase in [H(+)] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (approximately 10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK(a) values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.
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Dyson, H. J., & Beattie, J. K. (1982). Spin state and unfolding equilibria of ferricytochrome c in acidic solutions. J Biol Chem, 257(5), 2267–2273.
Abstract: Equilibrium, stopped flow, and temperature-jump spectrophotometry have been used to identify processes in the unfolding of ferricytochrome c in acidic aqueous solutions. A relaxation occurring in approximately 100 microseconds involves perturbation of a spin-equilibrium between two folded conformers of the protein with methionine-80 coordinated or dissociated from the heme iron. The protein unfolds more slowly, in milliseconds, with dissociation and protonation of histidine-18. These two transitions appear cooperative in equilibrium measurements at low (0.01 M) ionic strength, but are separated at higher (0.10 M) ionic strength. They are resolved under both conditions in the dynamic measurements. The spin-equilibrium description permits a unified explanation of a number of properties of ferricytochrome c in acidic aqueous solutions.
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Saigo, S. (1981). A transient spin-state change during alkaline isomerization of ferricytochrome c. J Biochem (Tokyo), 89(6), 1977–1980.
Abstract: Kinetic difference spectra during the alkaline isomerization of ferricytochrome c were obtained by the pH-jump method in the range of 540 to 655 nm. The spectrum of the transient intermediate, which appears during the course of the isomerization, was reproduced from the spectra. The intermediate showed an intense absorption band at 600 nm, indicating that it is a high spin or mixed spin species. This is in contrast to the stable neutral and alkaline forms which are low spin species. The transient spin-state change during the isomerization was also observed upon rapid oxidation of ferrocytochrome c at alkaline pH.
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Matzke, S. M., Oubre, J. L., Caranto, G. R., Gentry, M. K., & Galbicka, G. (1999). Behavioral and immunological effects of exogenous butyrylcholinesterase in rhesus monkeys. Pharmacol Biochem Behav, 62(3), 523–530.
Abstract: Although conventional therapies prevent organophosphate (OP) lethality, laboratory animals exposed to such treatments typically display behavioral incapacitation. Pretreatment with purified exogenous human or equine serum butyrylcholinesterase (Eq-BuChE), conversely, has effectively prevented OP lethality in rats and rhesus monkeys, without producing the adverse side effects associated with conventional treatments. In monkeys, however, using a commercial preparation of Eq-BuChE has been reported to incapacitate responding. In the present study, repeated administration of commercially prepared Eq-BuChE had no systematic effect on behavior in rhesus monkeys as measured by a six-item serial probe recognition task, despite 7- to 18-fold increases in baseline BuChE levels in blood. Antibody production induced by the enzyme was slight after the first injection and more pronounced following the second injection. The lack of behavioral effects, the relatively long in vivo half-life, and the previously demonstrated efficacy of BuChE as a biological scavenger for highly toxic OPs make BuChE potentially more effective than current treatment regimens for OP toxicity.
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Haruta, N., & Kitagawa, T. (2002). Time-resolved UV resonance Raman investigation of protein folding using a rapid mixer: characterization of kinetic folding intermediates of apomyoglobin. Biochemistry, 41(21), 6595–6604.
Abstract: The 244-nm excited transient UV resonance Raman spectra are observed for the refolding intermediates of horse apomyoglobin (h-apoMb) with a newly constructed mixed flow cell system, and the results are interpreted on the basis of the spectra observed for the equilibrium acid unfolding of the same protein. The dead time of mixing, which was determined with the appearance of UV Raman bands of imidazolium upon mixing of imidazole with acid, was 150 micros under the flow rate that was adopted. The pH-jump experiments of h-apoMb from pH 2.2 to 5.6 conducted with this device demonstrated the presence of three folding intermediates. On the basis of the analysis of W3 and W7 bands of Trp7 and Trp14, the first intermediate, formed before 250 micros, involved incorporation of Trp14 into the alpha-helix from a random coil. The frequency shift of the W3 band of Trp14 observed for this process was reproduced with a model peptide of the A helix when it forms the alpha-helix. In the second intermediate, formed around 1 ms after the start of refolding, the surroundings of both Trp7 and Trp14 were significantly hydrophobic, suggesting the formation of the hydrophobic core. In the third intermediate appearing around 3 ms, the hydrophobicity was relaxed to the same level as that of the pH 4 equilibrium intermediate, which was investigated in detail with the stationary state technique. The change from the third intermediate to the native state needs more time than 40 ms, while the appearance of the native spectrum after the mixing of the same solutions was confirmed separately.
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Ballew, R. M., Sabelko, J., & Gruebele, M. (1996). Direct observation of fast protein folding: the initial collapse of apomyoglobin. Proc. Natl. Acad. Sci. U.S.A., 93(12), 5759–5764.
Abstract: The rapid refolding dynamics of apomyoglobin are followed by a new temperature-jump fluorescence technique on a 15-ns to 0.5-ms time scale in vitro. The apparatus measures the protein-folding history in a single sweep in standard aqueous buffers. The earliest steps during folding to a compact state are observed and are complete in under 20 micros. Experiments on mutants and consideration of steady-state CD and fluorescence spectra indicate that the observed microsecond phase monitors assembly of an A x (H x G) helix subunit. Measurements at different viscosities indicate diffusive behavior even at low viscosities, in agreement with motions of a solvent-exposed protein during the initial collapse.
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Gulotta, M., Gilmanshin, R., Buscher, T. C., Callender, R. H., & Dyer, R. B. (2001). Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape. Biochemistry, 40(17), 5137–5143.
Abstract: An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.
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