In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms.
We carried out simulator studies on ceramic-polyethylene total-hip combinations to determine the volumetric wear-rates of 22 mm, 26 mm and 28 mm femoral-head sizes. Bovine-serum lubrication and 2 kN peak sinusoidal load-profile were used with polyethylene (UHMWPE) cups. Wear was assessed by gravimetric technique. Precision (9%) was ensured by the use of multiple specimens, multiple wear-events, and the linear-regression method of estimating the average wear trend, thereby reducing the inherent, unpredictable nature of each wear-event. Volumetric wear-rates for polyethylene averaged 23 mm3 per 10(6) cycles for the 22 mm ceramic head and up to 32 mm3 per 10(6) cycles for the 28 mm head. The difference between 22 mm and the larger head-sizes was significant. This may well be the first laboratory confirmation of Charnley's original clinical Low-Friction Arthroplasty concept with regard to wear rate. The wear penalty increased linearly at the rate of 6% to 9% per mm of diameter increase.
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