Abstract: We have here investigated the dissociation kinetics of the His side chains axially ligated to the heme-iron in the ferric (1-56 residues) N-fragment of horse cyt c. The ligand deligation induced by acidic pH-jump occurs as a biexponential process with different pre-exponential factors, consistent with a structural heterogeneity in solution and the presence of two differently coordinated species. In analogy with GuHCl-denatured cyt c, our data indicate the presence in solution of two ferric forms of the N-fragment characterized by bis-His coordination, as summarized in the following scheme: His18-Fe(III)-His26 <==> His18-Fe(III)-His33. We have found that the pre-exponential factors depend on the extent of the pH-jump. This may be correlated with the different pKa values shown by His26 and His33; due to steric factors, His26 binds to the heme-Fe(III) less strongly than His33, as recently shown by studies on denatured cyt c. Interestingly, the two lifetimes are affected by temperature but not by the extent of the pH-jump. The lower pKa for the deligation reaction required the use of an improved laser pH-jump setup, capable of inducing changes in H+ concentration as large as 1 mM after the end of the laser pulse. For the ferric N-fragment, close activation entropy values have been determined for the two histidines coordinated to the iron; this result significantly differs from that for GuHCl-denatured cyt c, where largely different values of activation entropy were calculated. This underlines the role played by the missing segment (residues 57-104) peptide chain in discriminating deligation of the “nonnative” His from the sixth coordination position of the metal.

Abstract: Growth of Rhodococcus equi was studied in vitro. Optimal growth occurred under aerobic conditions between pH 7.0 and 8.5, at 30 degrees C. R. equi survived better in a neutral soil (pH 7.3) than it did in two acid soils (pH less than 5.5). It grew substantially better in soils enriched with faeces than in soils alone. Simple organic acids in horse dung, especially acetate and propionate, appear to be important in supporting growth of R. equi in the environment. The ecology of R. equi can be best explained by an environmental cycle allowing its proliferation in dung, influenced by management, grazing behaviour and prevailing climatic conditions. Preventive measures should be aimed at reducing or avoiding focal areas of faecal contamination in the environment.

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: 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.