Bioluminescence of monolayers of firefly luciferase immobilized on graphite.

We report on the immobilization of the firefly protein luciferase on the hydrophobic surface of graphite. Observation by liquid-phase atomic force microscopy of islands with a height consistent with the size of a single molecule confirmed that the protein was contained within a monomolecular layer. The enzyme activity was assayed by single-photon counting of the bioluminescence, which is the catalytic product of luciferase. Attachment to the surface modified the efficiency of the enzyme, but the introduction of the substrates luciferin and ATP resulted in the reactivation of the enzyme. The functionalized graphite surface was employed as a cathode in a bioelectrochemical cell. This demonstrated that the electric field caused a substantial loss of enzyme catalytic activity.

Modulation of firefly luciferase bioluminescence at bioelectrochemical interfaces.

This paper describes a method by which the activity of an immobilized enzyme can be modulated electrochemically at an electrode. The particular example studied, involving the enzyme firefly luciferase being immobilized in a gelatin film of thickness <1 µm, provides a useful model system since changes in the catalytic activity can be measured instantaneously through the natural bioluminescent emission. Using this biointerfacial arrangement, we have been able to demonstrate the reversible switching off and on of the enzyme's activity. Through a series of mechanistic studies, we have been able to determine that the bioluminescence response is modulated (on long time scales) as a consequence of the electrochemical depletion of protons at the electrode interface resulting in a local increase in pH.

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.

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 first-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 microseconds 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.