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.

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.

Abstract: Chemical relaxation studies on the system horse liver alcohol dehydrogenase, nicotinamide adenine dinucleotide, and ethanol were conducted observing fluorescence changes between 400 and 500 nm. Temperature-jump experiments were performed at pH 6.5, 7.0, 8.0, and 9.0; concentration-jump experiments at pH 9.0. The reciprocal of the slowest relaxation time was found to be linearly dependent upon the enzyme concentration for relatively low enzyme concentrations, as predicted earlier. Use of the wide pH-range necessitated expression of the four apparent dissociation constants of the catalytic reaction cycle in terms of pH-independent constants. The system was described in terms of only one (or two) catalysis-linked protons not associated with the electron transfer. Protonic steps in a buffered system are in rapid equilibrium, too fast to be measured with the equipment available. Assuming only two of the four bimolecular reaction steps in the four-step cycle are fast compared to the remaining two, six cases may be considered with six expressions for the reciprocal of the slowest relaxation time. Comparison with the experimental data revealed that the bimolecular reaction steps governing the slowest relaxation time change with pH. Above the effective time resolution of the temperature-lump instrument with fluorescence detection (0.1 msec) only one other relaxation time was detectable and only at pH 9. This relaxation time, found to be independent of the concentration of all reactants within experimental error (r = 10 +/- 5 msec), is most likely due to an interconversion among ternary complexes.

Abstract: Several years ago, Theorell and Czerlinski conducted experiments on the system of horse liver alcohol dehydrogenase, reduced nicotinamide adenine dinucleotide and imidazole, using the first version of the temperature jump apparatus with detection of changes in fluorescence. These early experiments were repeated with improved instrumentation and confirmed the early experiments in general terms. However, the improved detection system allowed to measure a slight concentration dependence of the relaxation time of around 3 ms. Furthermore, the chemical relaxation time was smaller than the one determined earlier (by factor 2). The data were evaluated much more rigorously than before, allowing an appropriate interpretation of the results. The observed relaxation time is largely due to rate constants in an interconversion of ternary complexes, which are faster than three (of the four) dissociation rate constants, determined previously by Theorell and McKinley-McKee.1,2 This fact contributed to earlier difficulties of finding any concentration dependence. However, the binding of imidazole to the binary enzyme-coenzyme complex can be made to couple kinetically into the interconversion rate of the two ternary complexes. The observed signal derives largely from the ternary complex(es). A substantial fluorescence signal change is associated with the observed relaxation process, suggesting a relocation of the imidazole in reference to the nicotinamide moiety of the bound coenzyme. Nine models are considered with two types of coupling of pre-equilibria (none-all). Quantitative evaluations favor the model with two ternary complexes connected by an interconversion outside the four-step (bimolecular) cycle. The ternary complex outside the cycle has much higher fluorescence yield than the one inside. The interconversion equilibrium is near unity for imidazole. If it would be shifted very much to the side of the “dead-end” complex (as in isobutyramide?!), stimulating action could not take place.