No abstract

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.

Chromatin core particles containing 146 base pairs of DNA have been found to undergo a single defined transition below 10 mM ionic strength as studied by both sedimentation velocity and tyrosine fluorescence anisotropy. A method is described for the preparation of such core particles from chicken erythrocytes with greater than 50% yield.

We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H(+)], which can be more than 100 microM, is complete within a few nanoseconds. The increase in [H(+)] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (approximately 10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK(a) values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.

Comparisons of theoretical calculations and experimental results are reported for the anisotropy of the hyperfine splitting and g value of the di-t-butyl nitroxide free radical. Extensive experimental data were collected with a microwave frequency of 9.5 GHz with supplementary data collected at 35 GHz. Several approximations to the Hamiltonian are discussed and compared to the results of the exact solution. Explicit equations for several approximations are given in terms of the principal values of the splitting and g-value tensors and the orientation of the nitroxide in the external magnetic field. The more accurate approximations should prove useful in the interpretation of nitroxide ESR spectra by decreasing the programming difficulties and the time requirements involved in computer simulation of nitroxide spectra.

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.

No abstract

The low-salt transition of chicken erythrocyte core particles containing uniform 145 base pair DNA was studied as a function of pH and of salt concentration. Intrinsic tyrosine fluorescence was used to follow the changes. Potassium salts of the anions C1-, H2PO4-, and SO4(2-) were indistinguishable in their ability to affect the transition. Divalent cations (Mg2+, Mn2+, Ca2+) were effective at 36-fold lower total concentration than monovalent cations (Li+, Na+, K+, Tris+), but no significant differences were observed within the two classes of cations. These results indicate that cation binding to the core particle is involved in the transition. At pH 9 the transition was broadened and shifted to higher monovalent cation concentration as compared to that at pH 6. At both pHs the fluorescence changes could be resolved into two steps by numerical least-squares analysis. On the basis of what is known about histone--histone interactions, a two-step mechanism is suggested, involving changes in the interactions between dimers of histones 2a and 2b with a tetramer of histones 3 and 4. The pH-induced changes appear to be correlated with a structural transition, which was detected as a function of pH at near physiological ionic strength (0.1 M). This structural change was accompanied by a small decrease in the tyrosine fluorescence anisotropy. An apparent pKa value near 7 is indicated, suggesting that the structural changes involved may be of physiological significance.