Search PubMed⌕ Search

Biomedical subjects

K Kuwajima

Publications and source records attributed to K Kuwajima.

103 records · Page 6Linked to original sources
PubMed1978

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin.

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.

Circular Dichroism↗
PubMed1978

Equilibrium and kinetics of the thermal unfolding of alpha-lactalbumin. The relation to its folding mechanism.

The thermal unfolding of alpha-lactalbumin has been studied by equilibrium measurements of aromatic difference spectra, and by kinetic measurements of the Joule heating temperature-jump. The unfolding at neutral pH is a reversible two-state transition. The equilibrium transition curves are analyzed by the nonlinear squares method, which gives correct values of thermodynamic parameters based on the data in a wide range of temperature. The results are discussed in relation to the previous studies on the unfolding by guanidine hydrochloride or by acid. The thermally unfolded state, a partially unfolded species, is shown to be thermodynamically similar to but not identical with the acid state. The folding pathway deduced from the kinetic results is essentially consistent with the folding model of alpha-lactalbumin proposed previously. Large decreases in entropy and in heat capacity during the reversed activation suggest the packing of the folded segments by hydrophobic interactions, while the forward activation shows a marked temperature dependence, probably caused by the disruption of specific long-range interactions.

Chemical Phenomena↗
PubMed1977

Thermodynamic characterization of partially denatured states in the denaturation process of bovine alpha-lactalbumin by inorganic denaturants.

In an attempt to understand the specific effect of inorganic protein denaturants, lithium cation and perchlorate anion, upon the molecular conformation of bovine alpha-lactalbumin and to characterize the denatured states of the protein and the denaturation processes, themodynamic studies on the reversible unfolding of the protein in the presence of lithium chloride, lithium perchlorate and sodium perchlorate were made by means of circular dichroism and ultraviolet absorption measurements. The denaturation reaction caused by lithium chloride was found to take place in a three-state type, while that caused by the two perchlorates in a two-state type. The latter produces the same denatured state as the acid does on the protein, the state where the helical structures remain unchanged. The former produces two kinds of the denatured state, one being a less unfolded state than the acid denatured one and the other a fully unfolded state which is identical with the finally denatured state induced by organic denaturants such as guanidinium chloride, guanidinium thiocyanate and urea.

Animals↗
PubMed1977

Equilibrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (IV): dependence of the N equilibrium A transconformation on the temperature.

The reversible unfolding from the native (N) state to the acid (A) state of alpha-lactalbumin by guanidine-HCl (0.8-2.0 M) was studied at 10-35 degrees C by means of difference spectra and pH-jump measurements. At each temperature, all points plotted as the logarithmic equilibrium constant log KNA of the N equilibrium A process against pH could fall on a curve independent of the denaturant concentration by shifting each point along the log KNA axis, where the shift factor f did not depend on temperature. Moreover, by shifting the points at each temperature along the log (KNA/f) axis, a master curve, independent of both temperature and the denaturant concentration, could be obtained for the pH-dependence of log KNA. From the dependence of the logarithmic rate constants on pH, master curves independent of both temperature and the denaturant concentration could also be made for the N leads to A and the A leads to A processes, where A mean the activated state. The results show the two-state character of the N equilibrium A process. The enthalpy changes and the differences in heat capacity for the N equilibrium A, N equilibrium A and A equilibrium A processes were determined from the accurate measurements of temperature dependence of the unfolding at pH 4.3 and 1.0 M guanidine-HCl. The results show that the disruption of hydrophobic interaction is caused mainly in the A leads to A process, while most of the changes in the pK values of the ionizing groups are caused in the N leads to A process.

Guanidines↗
PubMed1976

Equilbrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (II).

The reversible unfolding of alpha-lactalbumin by guanidine hydrochloride, was studied at 25.0 degrees C in a relatively low concentration range of the denaturant (0.80-2.00 mol/l) by means of difference spectra and pH-jump measurements. The unfolding was shown to occur between two states, N and D, because apparent rate-constants of the unfolding and the refolding reactions depended only on pH. All curves plotted as the logarithmical equilibrium constant log KD against pH could fall on the same base curve by shifting each curve along the log KD axis. From the dependence of the logarithmic rate constant on pH, master curves could also be made for the forward and the backward reactions. The dependence of these master curves on pH indicates that the groups affecting the pH dependence of the unfolding are three residues with pKN = 3.3 and pKA = pKD = 4.4, one residue with pKN = pKA = 3.8 and pKD = 4.4, and one residue with pKN = 5.8 and pKA = pKD = 6.3, where A indicates the activated state. On the other hand, from the denaturant activity dependence of the shift factors required for making the master curves, the value of the intrinsic binding constant of the denaturant to the protein was found to be similar to that obtained from previous measurements at pH 5.5. Differences between the numbers of the binding sites of the denaturant on the denaturated and the native proteins, and between those on the activated and the native proteins were shown to be 5.3 and 2.1, respectively. The free energy of stabilization in the native-like environment also shows that the protein in the native state is more unstable than lysozyme.

Animals↗
PubMed1975

Electrophoretic investigations of the acid conformational change of alpha-lactalbumin.

In order to clarify how the electrophoretic behavior reflects the conformational transition of globular proteins, moving boundary electrophoresis was applied to analysis of the acid conformational change of alpha-lactalbumin. The appearance of only a single electrophoretic boundary in the transition region of the protein suggests a very rapid transition with a half-time estimated to be smaller than 7 min on the basis of the theory of isomerizing systems in electrophoresis. The transition is clearly reflected in the dependence of the mobility on the protein net charge, which shows a sigmoidal curve closely similar to that obtained by a Linderstrøm-Lang pH-tritration plot for the carboxyl groups of alpha-lactalbumin. It was also concluded from the transition curves that the acidfication does not result in complete unfolding, but that a compact structure is maintained in the acidic region with an apparently expanded form as compared to the native state of the protein. All results obtained by electrophoresis were also supported by the results of pH-jump studies, analytical gel chromatography, and CD measurements.

Circular Dichroism↗