Abstract: Association equilibria and association kinetics of the thiocyanate binding reaction to methemoglobin in single crystals and solution are studied using temperature-jump technique and polarized absorption spectroscopy. Different kinetic constants are found for the reaction in solution and crystal phase for the alpha- and beta-subunits of the methemoglobin tetramer. The reduction of the reactivity of the alpha- and beta-subunits in crystalline phase is 6-fold and 2.4-fold, respectively, compared to the values found in solution. The intramolecular binding reaction of the N epsilon of the distal histidine E7 which is observed in methemoglobin in solution cannot be detected in single crystals. Our results suggest that crystallization of hemoglobin has little influence on small-scale structural fluctuations which are necessary for ligands to get to the binding sites and large-scale structural motions are suppressed.

Abstract: Temperature-dependent EPR and temperature-jump measurements have been carried out, in order to examine the high-spin to low-spin transition of aquomethemogobin (pH 6.0). Relaxation rates and equilibrium constants could be determined as a function of temperature. As a reaction mechanism for the high-spin to low-spin transition, the binding of N epsilon of His E7 to the heme iron had been proposed; the same mechanism had been suggested for the ms-effect, found in temperature-jump experiments on aquomethemoglobin. A comparison of the thermodynamic quantities, deduced form the measurements in this paper, gives evidence that indeed the same reaction is investigated in both cases. Our results and most of the findings of earlier studies on the spin-state transitions of aquomethemoglobin, using susceptibility, optical, or EPR measurements, can be explained by the transition of methemoglobin with H2O as ligand (with high-spin state at all temperatures) and methemoglobin with ligand N epsilon of His E7 (with a low-spin ground state). Thermal fluctuations of large amplitude have to be postulated for the reaction to take place, so this reaction may be understood as a probe for the study of protein dynamics.

Abstract: The dynamic behavior of various types of cytochromes c in the redox reaction with iron hexacyanides was studied using a temperature-jump method in order to elucidate the molecular mechanism of the redox reaction of cytochromes with their oxidoreductants. Transmittance after the temperature jump changed through a single exponential decay for all cytochromes investigated. Under a constant concentration of anion, the redox reaction of various types of cytochrome c with iron hexacyanides was analyzed according to the scheme: Ki=kt/k-i (i=1,2,3) where C(III) and C(II) are ferric and ferrous cytochromes, respectively, Fe(III) and Fe(II) are ferri- and ferrocyanides, respectively, C(III) [middle dot] Fe(II) is the ferricytochrome-ferrocyanide complex and C(II) [middle dot] Fe(III) is the ferrocytochrome-ferricyanide complex. When step B is slower than the other two steps A and C, τ-1 can be represented approximately as where the bar over the variables denotes the equilibrium value. In a large excess of ferrocyanide against cytochrome, we can estimate k2, k-2, K1 and K3 independently. In the case of horse cytochrome c at 18[degree sign]C in 0.1 M phosphate buffer at pH 7 with 0.3 M KNO3, the estimated parameters are k2 = 100 +/- 50 s-1, k-2 = (3.5 +/- 1.0) [middle dot] 103 s-1, K1 = 15 +/- 7 M-1 and K3 = (8.5 +/- 1.5) [middle dot] 10-4 M. From the same experiments for seven cytochromes (cytochrome c from horse, tuna, Candida krusei, Saccharomyces oviformis, Rhodospirillum rubrum cytochrome c2, Spirulina platensis cytochrome c-554 and Thermus thermophilus cytochrome c-552), the following results can be deduced. (1) Each parameter defined in the scheme above (k2, k-2, K1, K3) diverged beyond the error range. Above all, k2 values of cytochromes c-554 and c-552 are as large as 1 [middle dot] 104 s-1 and much larger than those for the other cytochromes (to 50 approx. 700 S-1). (2) The variance of k2K1 and k-2/K3 are relatively less than the variances of individual parameters (k2, k-2, K1 and K3), which suggests that the values of k2K1 and k-2/K3 have been conserved during the course of evolution.

Abstract: The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers is continuous and multi-phasic. We have used a laser temperature-jump with fluorescence spectroscopy to measure the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.

Abstract: Many large mammals show pronounced seasonal fluctuations of metabolic rate (MR). It has been argued, based on studies in ruminants, that this variation merely results from different levels of locomotor activity (LA), and heat increment of feeding (HI). However, a recent study in red deer (Cervus elaphus) identified a previously unknown mechanism in ungulates--nocturnal hypometabolism--that contributed significantly to reduced energy expenditure, mainly during late winter. The relative contribution of these different mechanisms to seasonal adjustments of MR is still unknown, however. Therefore, in the study presented here we quantified for the first time the independent contribution of thermoregulation, LA and HI to heart rate (f(H)) as a measure of MR in a free-roaming large ungulate, the Przewalski horse or Takhi (Equus ferus przewalskii Poljakow). f(H) varied periodically throughout the year with a twofold increase from a mean of 44 beats min(-1) during December and January to a spring peak of 89 beats min(-1) at the beginning of May. LA increased from 23% per day during December and January to a mean level of 53% per day during May, and declined again thereafter. Daily mean subcutaneous body temperature (T(s)) declined continuously during winter and reached a nadir at the beginning of April (annual range was 5.8 degrees C), well after the annual low of air temperature and LA. Lower T(s) during winter contributed considerably to the reduction in f(H). In addition to thermoregulation, f(H) was affected by reproduction, LA, HI and unexplained seasonal variation, presumably reflecting to some degree changes in organ mass. The observed phase relations of seasonal changes indicate that energy expenditure was not a consequence of energy uptake but is under endogenous control, preparing the organism well in advance of seasonal energetic demands.