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...

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.

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 isomerization of horse-heart ferricytochrome c caused by varying pH was kinetically studied by using circular dichroism (CD) and optical absorption stopped-flow techniques. In the pH range of 7--13, the existence of the three different forms of ferricytochrome c (pH less than 10, pH 10--12, and pH greater than 12) was indicated from the statistical difference CD spectra. On the basis of analyses of the stopped-flow traces in the near-ultraviolet and Soret wavelength regions, the isomerization of ferricytochrome c from neutral form to the above three alkaline forms was interpreted as follows (1) below pH 10, the replacement of the intrinsic ligand of methionine residue by lysine residue occurs; (2) between pH 10 and 12, the uncoupling of the polypeptide chain from close proximity of the heme group occurs first, followed by the interconversion of the intrinsic ligands; and (3) above pH 12, hydroxide form of ferricytochrome c is formed, though the replacement of the intrinsic ligand by extrinsic ligands may occur via different routes from those below pH 12. The CD changes at 288 nm and in the Soret region caused by the pH-jump (down) from pH 6.0 to 1.6 were compared with the appearance of the 620-nm absorption band ascribed to the formation of the high-spin form of ferricytochrome c. Both CD and absorption changes indicated that the isomerization at pH 1.6 consisted of two processes: one proceeded within the dead-time (about 2 ms) of the stopped-flow apparatus and the other proceeded at a determinable rate with the apparatus. On the basis of these results, the isomerization of ferricytochrome c at pH 1.6 was explained as follows: (1) the transition from the low-spin form to the high-spin forms occurs within about 2 ms, the dead-time of the stopped-flow apparatus; and (2) the polypeptide chain is unfolded after the formation of the high-spin form.