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.

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.

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: Horses are known to show fear reactions when confronted with novelty and this can be a considerable hindrance in the context of working situations such as riding, dressage or racing. The aim of the present study was to measure the potential effects of a synthetic analogue of the Equine Appeasing Pheromone on saddled horses when subjected to a stressful situation using a double-blinded, placebo controlled study design. A group of 40 horses was analyzed during this study and horses were divided by sex, breed and reactivity into two homogenized groups. The test, which consisted of walking the horse through a fringed curtain, was selected from a range of tests which are used to assess behaviour for the selection of French breeding stock. Horses that could have been subjected to the test on a previous occasion, and therefore be familiar with it, were not included. Behavioural and physiological parameters were both taken into account with measures of time to go through the curtain, fear related typical behavioural patterns, based on available literature detailed in the bibliography, and heart rate being recorded. Parameters were analyzed by means of Mann-Whitney U-test. Significant differences were noticed between the two groups concerning heart rate data during the test (UMeanHR = 100.5, pMeanHR = 0.02; UMaxHR = 75, pMaxHR = 0.001) and during the whole measured period (UMeanHR = 67, pMeanHR = 0.005; UMaxHR = 58, pMaxHR = 0.002). Observation of the animals also revealed less behavioural items characteristic of fear within the treated group. As a result, horses performed the test with a better time performance when they received the pheromone analogue (U = 62, p = 0.002). The main parameter, area under the HR graph, is based on heart rate measure and performance. Differences noticed (U = 74, p = 0.002) for this parameter lead to the conclusion that horses who received EAP underwent less stress related consequences in terms of their cardiac physiology. As horses are subjected to a number of foreseeable stressful events this study suggests that the use of Equine Appeasing Pheromone could be a significant factor in improving the welfare of this species.