Comparative study of the pressure effects on the magnetic penetration depth in electron- and hole-doped cuprate superconductors.

The effect of pressure on the magnetic penetration depth λ was tested for the hole-doped superconductor YBa(2)Cu(3)O(7-δ) and in the electron-doped one Sr(0.9)La(0.1)CuO(2) by means of magnetization measurements. Whereas a large change of λ was found in YBa(2)Cu(3)O(7-δ), confirming the non-adiabatic character of the electron-phonon coupling in hole-doped superconductors, the same quantity is not affected by pressure in electron-doped Sr(0.9)La(0.1)CuO(2), suggesting a close similarity of the latter to conventional adiabatic Bardeen-Cooper-Schrieffer superconductors. The present results imply a remarkable difference between the electronic properties of hole-doped cuprates and electron-doped Sr(0.9)La(0.1)CuO(2), giving a strong contribution to the long debated asymmetric consequences of hole and electron doping in cuprate superconductors.

Effect-site concentration of remifentanil for laryngeal mask airway insertion during target-controlled infusion of propofol.

The purpose of this study was to determine the effect-site concentration of remifentanil that would provide optimal conditions for successful laryngeal mask airway insertion during a target-controlled infusion (TCI) of propofol at 3.5 microg.ml(-1) without the use of neuromuscular blockade. Five minutes after propofol infusion, remifentanil was infused at a dose determined by a modified Dixon's up-and-down method. Five minutes after remifentanil infusion, the laryngeal mask was inserted. The effect-site concentration of remifentanil for successful laryngeal mask insertion in 50% of adults (EC(50)) was 3.04 (SD 0.49) ng.ml(-1) during a TCI of 3.5 microg.ml(-1) propofol without neuromuscular blockade. From the probit analysis, the EC(50) and EC(95) of remifentanil were 2.84 ng.ml(-1) (95% CI 2.09-3.57 ng.ml(-1)) and 3.79 ng.ml(-1) (95% CI 3.26-9.25 ng.ml(-1)), respectively.

Size and fractal dimension of particles in the River Nakdong.

Water samples were collected at seven sites located along the River Nakdong on 30 occasions. Water quality, size and the fractal dimension (dF) of suspended particles were measured. The laser light scattering method was used to obtain the size and dF of suspended particles. The average size of particles in this river ranged from 89 microm and 169 microm, which appears to be relatively coarse compared with other rivers worldwide. The average dF of suspended particles in this study ranged from 1.8 to 1.9. Slight variations in fractal dimension values and other particle characteristics results from various measuring methods available. The correlation analysis showed that DO, TN, NO3 and chlorophyll-a had significant positive relationships with particles size, whereas flow rates and temperature had negative relationships. However, the factors which had positive relationships with particles size showed negative relationships with the dF of suspended particles. Generally, as the size of particles increased, the fractal dimension of particles decreased which indicated that the shape of the larger particles became more irregular relative to that of the smaller ones. To obtain and apply the statistical functional relationship between water quality characteristics, multiple linear regression equations of the size and fractal dimension of particles on explanatory variables such as pH, BOD, TSS, DO, T-N and T-P have been established.

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin.

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

Cation-binding sites of subtilisin Carlsberg probed with Eu(III) luminescence.

Two Ca(2+)-binding sites of subtilisin Carlsberg are studied by monitoring static and time-resolved luminescence of selectively substituted Eu(3+) at each site, and they are found to be characteristically quite different from each other. Compared with the coordination sphere of free Eu(3+), two sites are very similar to each other, so that both have a well-defined binding structure with low coordination symmetry. However, compared with the weak site, the strong site is relatively more polar, more symmetrical, and more easily accessible. Furthermore, despite the absence of water reported in the x-ray crystal structure (, Eur. J. Biochem. 166:673-692), one water molecule is found to exist in the coordination sphere of the strong site in aqueous solution. Thus it is suggested that in solution the Ca(2+) bound in the strong site forms an additional coordination bond to a solvent or substrate molecule.

Progressive rearrangement of subtilisin Carlsberg into orderly and inflexible conformation with Ca(2+) binding.

Fluorescence depolarization and decay kinetic profiles, together with differential scanning calorimetric thermograms and circular dichroism spectra, are measured to understand the respective roles of Ca(2+) ions at the strong (Ca1) and weak binding sites (Ca2) of subtilisin Carlsberg (sC). Thermal denaturation temperature decreases considerably with Ca1 removal, whereas it does slightly with Ca2 removal. The fraction of random coil structure increases significantly with Ca2 removal as well as with Ca1 removal. sC shows three fluorescence decay times of 100, 1100, and 3300 ps. Although the fast and the slow do not change noticeably, the medium one decreases progressively with Ca(2+) removal. sC has two fluorescence anisotropic decay components of 340 and 12,000 ps. The fast one arises from the internal rotation of Tyr, whereas the slow results from the global rotation of sC. Although both become significantly faster with Ca2 removal, only the slow one becomes slightly faster with further Ca1 removal. Overall, sC undergoes progressive rearrangement into disorderly and flexible conformation with Ca(2+) removal, indicating that both Ca1 and Ca2 are indispensable for the stable structure of sC.

Deprotonation of lipid-depleted bacteriorhodopsin.

The removal of 75% of the lipid from bacteriorhodopsin caused the following: (i) decreased efficiency and rate of deprotonation of the protonated Schiff base (as monitored by absorption of the M412 intermediate); (ii) increased efficiency of deprotonation of deionized samples; (iii) a decrease by 1 unit in the pH at which deprotonation ceases; (iv) increased intensity of Eu3+ emission in Eu3+-regenerated deionized delipidated samples; (v) increased exposure of the Eu3+ sites to water; and (vi) elimination of the dependence of the deprotonation efficiency on the metal cation concentration. These results are discussed in terms of changes in the protein conformation upon delipidation, which in turn control the deprotonation mechanism.

Tryptophan fluorescence quenching as a monitor for the protein conformation changes occurring during the photocycle of bacteriorhodopsin under different perturbations.

The rates of the quenching and recovery of tryptophan fluorescence are determined in the microsecond-millisecond time scale during the photocycle of bacteriorhodopsin under different perturbations. The kinetics suggest the presence of two quenching processes, a rapid one (on the time scale of photocycle intermediate L550 formation or faster) and a slow one (slightly slower than the slow component of intermediate M412 formation). The slow quenching process is found to respond to different perturbations in the same manner as the slow component of M412 formation. It has the same activation energy, it is inhibited if metal cations are removed, it is negligible at pH values greater than the pKa of tyrosine, and its rate is slowed down when 75% of the lipids are removed. These results, together with the observed value of the quenching activation energy, suggest that the rates of the tryptophan fluorescence quenching, like those of tyrosinate and M412 formations during the cycle, are all determined by the rates of the protein conformation changes. The pH studies of the slow quenching process show that the maximum quenching probability occurs at neutral pH. A rapid decrease in quenching occurs at lower pH (approximately 3 and approximately 5.5) and higher pH (approximately 9). Two quenching mechanisms involving energy transfer to either retinal or to tyrosinate are considered. Protein conformation changes resulting from a change in the ionization state of amino acids of different pKa values could change the tryptophan-retinal (or tryptophan-tyrosinate) coupling and thus the quenching efficiency.

Decay of the tryptophan fluorescence anisotropy in bacteriorhodopsin and its modified forms.

In this work we study the decay of the polarization of the Trp fluorescence in native bacteriorhodopsin (bR), deionized bR (dlbR), and the retinal-free form of bR, bacterioopsin (bO), using picosecond laser/streak camera system. Two types of depolarization processes are observed, one around 250 ps, which is temperature independent around room temperature, and the other in the 1-3-ns range, which is sensitive to temperature and certain bR modifications. This suggests the presence of at least two different environments for the eight Trp molecules in bR. Native bR and deionized bR gave the same depolarization decay times, suggesting that the removal of metal cations does not change the microenvironment of the emitting Trp molecules. The slow component is faster in bO than in bR, suggesting a change in the environment of the Trp molecules upon the removal of the retinal chromophore. All these results are discussed in terms of the different mechanisms of Trp fluorescence depolarization. A comparison between the depolarization decay in rhodopsin and bR is made.

Electron transfer between cytochrome a and copper A in cytochrome c oxidase: a perturbed equilibrium study.

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. We have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser (0.6-ns, 1.0-mJ pulses) to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 +/- 2000 s-1 (1 sigma), at pH 7.0 and 25.5 degrees C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome a and copper A is far faster than any rate measured or inferred previously for this process.

Effect of genetic modification of tyrosine-185 on the proton pump and the blue-to-purple transition in bacteriorhodopsin.

The retinylidene chromophore mutant (Y185F) of bacteriorhodopsin, in which Tyr-185 is substituted by phenylalanine, is examined and compared with wild-type bacteriorhodopsin expressed in Escherichia coli; both were reinstituted similarly in vesicles. The Y185F mutant shows (at least) two distinct spectra at neutral pH. Upon light absorption, the blue species (which absorbs in the red) behaves as if "dead"--i.e., neither its tyrosine nor its protonated Schiff base undergoes deprotonation nor does its tryptophan fluorescence undergo quenching. This result is unlike either the purple species (which absorbs in the blue) or wild-type bacteriorhodopsin expressed in E. coli. As the pH increases, both the color changes and the protonated Schiff base deprotonation efficiency suggest a blue-to-purple transition of the Y185F mutant near pH 9. If this blue-to-purple transition of Y185F corresponds to the blue-to-purple transition of purple-membrane (native) bacteriorhodopsin (occurring at pH 2.6) and of wild-type bacteriorhodopsin expressed in E. coli (occurring at pH 5), the protein-conformation changes of this transition as well as the protonated Schiff base deprotonation may be controlled not by surface pH alone, but rather by the coupling between surface potential and the general protein internal structure around the active site. The results also suggest that Tyr-185 does not deprotonate during the photocycle in purple-membrane bacteriorhodopsin.