N. Berovic

Optical diffraction by well-ordered muscle fibres

We have studied the diffraction of a focussed laser beam by single fibres of glycerinated rabbit psoas muscle as a function of the angle of incidence. Diffraction efficiencies as high as 34% were observed at the firs-order Bragg angle, indicative of well-ordered striated fibres with a strong periodic modulation of the refractive index. A theory is presented to account for our observations based upon the coupled-wave model developed by Kogelnik (1967) and Magnusson and Gaylord (1977) for the description of thick phase gratings in holography. We have solved the coupled-wave equations on a computer using a realistic index modulation taken from the measurements of Huxley and Hanson (1957). Comparison of theory with experiment shows that coupled-wave effects are indeed present in well-ordered muscle fibres, and the observed diffraction efficiency is in quite good agreement with what would be expected theoretically. Most importantly, the computer model allows us to calculate the diffraction efficiency for curved striations, which are observed for real muscle fibres under a microscope. The sensitivity of the diffraction efficiency to curvature of the striations may have implications for the interpretation of other optical experiments on muscle. We also consider the effects on our measurements of the focussing lens and refraction by the cylindrical fibre.

Observation of Brillouin scattering from single muscle fibres

The propagation of sound waves along relaxed single fibres of glycerinated rabbit psoas muscle has been observed using Brillouin scattering at frequencies up to 1.6 GHz. Two types of waves were observed: one with a velocity of 1508±7 m s−1, which is attributed to sound waves in intra-cellular saline, the other with a velocity of 912±25 m s−1, which is attributed to waves propagating along the protein filaments within individual sarcomeres. The latter sound velocity is much higher than that which has been reported by Stienen and Blangé (1985) for 50 μs tension transients, and the difference is attributed to the much higher stiffness of the protein filaments compared to the cross-bridges which determine the low-frequency elasticity of muscle fibres.

The Kinetics of Radiation Damage to the Protein Luciferase and Recovery of Enzyme Activity after Irradiation

Abstract Berovic, N., Pratontep, S., Bryant, A., Montouris, A. and Green, R. G. The Kinetics of Radiation Damage to the Protein Luciferase and Recovery of Enzyme Activity after Irradiation. Radiat. Res. 157, 122–127 (2002). Experimental observations are reported which follow the bioluminescence intensity of luciferase during irradiation by a 5 MeV proton beam. Bioluminescence is a measure of the protein enzyme activity and provides an assay of the enzyme rate of reaction in real time. Transient responses after a pulse of protons show recovery of the reaction rate with two time constants of 0.3 s−1 and 0.01 s−1. Changes in the reaction rate are due to radiation damage to the active form of the protein luciferase. Quantitative analysis of the radiation damage and recovery of the protein shows that products of the radiolysis of water play major part in the process of enzyme damage at room temperature. A few minutes after the pulse of protons, most of the enzyme activity has recovered. We attribute the fast recovery to the removal of charged ions, while the slow recovery involves refolding of denatured protein.

A critical analysis of the use of radiation inactivation to measure the mass of protein.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.

The quantum yield of luciferase is dependent on ATP and enzyme concentrations

Abstract The Quantum Yield of firefly luciferase relative to its substrate luciferin is a function of enzyme and ATP concentrations. The implications for molecular electronic applications are discussed.

Optical Switching and Amplification Based on the Enzyme Luciferase

Abstract Films containing luciferase and its substrates were made to act as optical memories and correlators.

Micrometre-scale bioluminescent enzyme photopatterning for bioelectronics applications

Abstract The enzyme firefly luciferase was patterned onto gold and glass surfaces at micrometre resolution using a novel photopatterning technique. The bioluminescence emitted on addition of the enzyme substrates luciferin and ATP allows these patterns to be visualised. Electrical switching of enzyme activity has been achieved by using enzyme immobilised on a gold surface as one electrode in an electrolysis cell. These techniques have applications in biosensor construction and in bioelectronics.