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Hillidge, C. J., & Lees, P. (1975). Cardiac output in the conscious and anaesthetised horse. Equine Vet J, 7(1), 16–21.
Abstract: Cardiac output in the horse was measured before and at predetermined times during 2-hour periods of thiopentone-halothane and thiopentone-diethyl ether anaesthesia. Left ventricular stroke volume was decreased to a similar extent during anaesthesia with each volatile agent, but a greater reduction in cardiac output occurred during halothane anaesthesia. This finding reflected the differing effects of halothane and ether on heart rate, a slight bradycardia occurring with the former agent while ether produced a small degree of tachycardia. The latter effect was attributed to enhanced sympathoadrenal activity. Changes in cardiac output and stroke volume were considered in relation to other factors, including arterial blood pH and tensions of oxygen and carbon dioxide. Positive correlations between some of these variables and cardiac function were established. With both volatile agents the reductions in stroke volume and cardiac output were related to the duration of anaesthesia, being greatest during the early stages. Possible reasons for the tendency of stroke volume and cardiac output to return towards control levels are discussed.
Keywords: Anesthesia, Inhalation/*veterinary; Animals; Carbon Dioxide/blood; *Cardiac Output/veterinary; *Consciousness; Electrocardiography/veterinary; Ether, Ethyl; Female; Halothane; Heart Rate; Heart Ventricles/physiology; Horses/*physiology; Hydrogen-Ion Concentration; Male; Oxygen/blood; Posture
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Jablonska, E. M., Ziolkowska, S. M., Gill, J., Szykula, R., & Faff, J. (1991). Changes in some haematological and metabolic indices in young horses during the first year of jump-training. Equine Vet J, 23(4), 309–311.
Abstract: Effects of an 18 min exercise test, on three separate occasions during a one year jump-training programme, was studied in seven horses. Determinations were carried out on venous blood for packed cell volume, haemoglobin, total protein, lactate and pyruvate, glucose, free fatty acids, insulin, glucagon, blood gases, bicarbonate, pH, aldolase, aspartate aminotransferase and alanine amino-transferase. Exercise caused a slight increase in lactate and pyruvate, total protein, aldolase, alanine aminotransferase, pO2, bicarbonate and pH. Glucose, free fatty acids and pCO2 levels decreased. Training caused no significant difference in these changes. However, during the year, increases in lactate and decreases in pH (resting levels) were observed.
Keywords: Alanine Transaminase/blood; Animals; Bicarbonates/blood; Blood Glucose/analysis; Blood Proteins/analysis; Breeding; Carbon Dioxide/blood; Exercise Test/veterinary; Fatty Acids, Nonesterified/blood; Female; Fructose-Bisphosphate Aldolase/blood; Hematocrit/veterinary; Hemoglobins/analysis; Horses/*blood/metabolism; Hydrogen-Ion Concentration; Lactates/blood; Male; Oxygen/blood; *Physical Conditioning, Animal; Pyruvates/blood
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Hinchcliff, K. W., Kohn, C. W., Geor, R., McCutcheon, L. J., Foreman, J., Andrews, F. M., et al. (1995). Acid:base and serum biochemistry changes in horses competing at a modified 1 Star 3-day-event. Equine Vet J Suppl, (20), 105–110.
Abstract: We examined the effects of participation in each of 3 modifications of Day 2 of a 3-day-event on blood and serum variables indicative of hydration, acid:base status and electrolyte homeostasis of horses. Three groups of horses – 8 European (E) horses and 2 groups each of 9 North American horses performed identical Days 1 (dressage) and 3 (stadium jumping) of a 3-day-event. E horses and one group of the North American horses (TD) performed modifications of Day 2 of a 1 Star 3-day-event and the other group of North American horses (HT) performed a Horse Trial on Day 2. Jugular venous blood was collected from each horse on the morning of Day 2 before any warm-up activity, between 4 min 55 s and 5 min 15 s after Phase D and the following morning. Eight E horses, 5 TD horses and 8 HT horses completed the trials. There were few significant differences in acid:base or serum biochemistry variables detected among horses performing either 2 variations of the Speed and Endurance day of a 1 Star 3-day-event, or a conventional Horse Trial. Failure to detect differences among groups may have been related to the low statistical power associated with the small number of horses, especially in the TD group, variation in quality of horses among groups and the different times of the day at which the E horses competed. Differences detected among time points were usually common to all groups and demonstrated metabolic acidosis with a compensatory respiratory alkalosis, a reduction in total body water and cation content, and hypocalcaemia. Importantly, horses of all groups did not replenish cation, chloride, and calcium deficits after 14-18 h of recovery.
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Rodier, F. (1976). [Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)]. Eur J Biochem, 63(2), 553–562.
Abstract: The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores...
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Andersson, P., Kvassman, J., Lindstrom, A., Olden, B., & Pettersson, G. (1981). Effect of NADH on the pKa of zinc-bound water in liver alcohol dehydrogenase. Eur J Biochem, 113(3), 425–433.
Abstract: Equilibrium constants for coenzyme binding to liver alcohol dehydrogenase have been determined over the pH range 10--12 by pH-jump stop-flow techniques. The binding of NADH or NAD+ requires the protonated form of an ionizing group (distinct from zinc-bound water) with a pKa of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a pKa of 11.2. The binding of trans-N,N-dimethylaminocinnamaldehyde to the enzyme . NADH complex is prevented by ionization of the latter group. It is concluded from these results that the pKa-11.2-dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic zinc ion of the enzyme subunit. The pKa value of 11.2 thus assigned to zinc-bound water in the enzyme . NADH complex appears to be typical for an aquo ligand in the inner-sphere ligand field provided by the zinc-binding amino acid residues in liver alcohol dehydrogenase. This means that the pKa of metal-bound water in zinc-containing enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active-site zinc ion.
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Wilson, M. T., Ranson, R. J., Masiakowski, P., Czarnecka, E., & Brunori, M. (1977). A kinetic study of the pH-dependent properties of the ferric undecapeptide of cytochrome c (microperoxidase). Eur J Biochem, 77(1), 193–199.
Abstract: The ferric form of the haem undecapeptide, derived from horse cytochrome c by peptic digestion, undergoes at least three pH-induced transitions with pK values of 3.4, 5.8 and 7.6. Temperature-jump experiments suggest that the first of these is due to the binding of a deprotonated imidazole group to the feric iron while the second and third arise from the binding of the two available amino groups present (the alpha-NH2 of valine and the epsilon-NH2 of lysine). Molecular models indicate that steric retraints on the peptide dictate that these amino groups may only coordinate to iron atoms via intermolecular bonds, thus leading to the polymerization of the peptide. Cyanide binding studies are in agreement with these conclusions and also yield a value of 3.6 X 10(6) M-1 s-1 for the intrinsic combination constant of CN- anion with the haem. A model is proposed which describes the pH-dependent properties of the ferric undecapeptide.
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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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Hoang, L., Maity, H., Krishna, M. M. G., Lin, Y., & Englander, S. W. (2003). Folding units govern the cytochrome c alkaline transition. J Mol Biol, 331(1), 37–43.
Abstract: The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.
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Pierce, M. M., & Nall, B. T. (2000). Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization. J Mol Biol, 298(5), 955–969.
Abstract: The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.
Keywords: Amino Acid Sequence; Amino Acid Substitution/genetics; Binding Sites; Cytochrome c Group/*chemistry/genetics/*metabolism; *Cytochromes c; Enzyme Stability/drug effects; Fluorescence; Guanidine/pharmacology; Heme/*metabolism; Histidine/genetics/*metabolism; Hydrogen-Ion Concentration; Isomerism; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation/genetics; Proline/*chemistry/metabolism; Protein Conformation/drug effects; Protein Denaturation/drug effects; *Protein Folding; Protein Renaturation; Saccharomyces cerevisiae/enzymology/genetics; Sequence Alignment; Thermodynamics
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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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