NAD+ kinase: molecular weight determination by low-angle laser-light scattering

Low-angle laser-light scattering (LALLS) was employed to measure the absolute molecular weight of chicken liver NAD+ kinase (NADK). The weight-average molecular weight (Mw) was found to be 275 000 +/- 15 000. The corresponding value for the second virial coefficient was -1.65 X 10(-3) ml X mol X g2. The value for Mw is in close accord with estimates reported for pigeon liver (270 000) and C. utilis (260 000) NADK. If the active enzyme is a dimer, the weight difference between pigeon/chicken liver and rabbit liver (136 000) NADK would indicate that the latter enzyme is an active monomer unit.     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

Characterization and hydrodynamic behaviour of modified gelatin: I. Light scattering and viscometry studies

Commercial samples of gelatin modified by succinylation and currently used as plasma substitutes and fractionated samples obtained by diafiltration have been studied by viscometry, light scattering and osmometry. Viscometric results show that the aqueous medium containing potassium phosphate (0.1 ) and NaCl (0.12 ) at pH 3.3 behaves nearly like a theta solvent (a=0.48) for these modified gelatins. The Stockmayer-Fixman diagram reveals a negative slope attributed to a swelling of the macromolecules which decreases as the molecular weight _w increases. The Stokes radius R_H determined by quasielastic light scattering is independent of the pH of the medium in a range 7-3.3. The conformation of gelatins in solution has been characterized through the ratio _G· _H⁻¹, the radius of gyration _G being determined by viscometry. This ratio decreases as the molecular weight increases. The low molecular weight fractions have a more compact structure than the Gaussian chains in theta conditions. For high molecuar weight fractions, the values of _G· _H⁻¹ tend to those of an hard sphere.     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration     

Effects of suspensoids (turbidity) on penetration of solar radiation in aquatic ecosystems

In mainland Australia and in southern Africa, the aridity of the climate and sparse vegetative cover increase the susceptibility of the soils to erosion, and as a consequence surface waters are usually turbid. The inanimate suspensoids in such waters, the tripton fraction of the limnologist, are responsible for virtually all the light scattering, and also, by virtue of the yellow-brown humic materials adsorbed on their surface, for a substantial part of the light absorption. Spectral absorption data for suspensoids in terms of theirin situ absorption coefficient values, and the contribution of suspensoids to absorption of photosynthetically available radiation (PAR) are given for certain Australian water bodies.To understand the effect of suspensoids on attenuation of the solar flux with depth, the scattering coefficient must also be known, and this can be determined from the nephelometric turbidity or from up- and down-welling irradiance measurements. The effect of particle size on scattering efficiency is discussed.An equation expressing the vertical attenuation coefficient for downward irradiance as a function of absorption coefficient, scattering coefficient and solar altitude is presented, and is used to explore the effects of absorption due to dissolved colour and suspensoids, and the effects of scattering by suspensoids, on the penetration of PAR.Suspensoids, by increasing the rate of attenuation of the solar flux with depth, can greatly diminish the euphotic depth of a water body, with a consequent decrease in the ratio of the euphotic to the mixed depth: thus turbidity can reduce productivity of a water body substantially below that which might be expected on the basis of nutrient availability. Shallow turbid waters of low intrinsic colour can, however, be highly productive. By diminishing the depth of the layer within which solar energy is dissipated as heat, suspensoids can greatly modify the hydrodynamic behaviour of water bodies, and this also has far-reaching ecological consequences.Suspensoids drastically impair the visual clarity of water, a fact of major significance for the aquatic fauna, as well of aesthetic significance for humanity. The reciprocal of the Secchi depth is more correctly thought of as a guide to the vertical contrast attenuation coefficient rather than to the vertical attenuation coefficient for irradiance. The reflectivity of a water body, being at any wavelength proportional to the backscattering coefficient divided by the absorption coefficient, is highly dependent on the concentration, and optical character, of the suspensoids present. This has implications not only for the appearance (colour, muddiness) of the water to an observer, but also for the remote sensing of water composition by air- or satellite-borne radiometric sensors.     

Second virial coefficient of alpha-crystallin

Light scattering studies were performed on bovine alpha-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of alpha-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of alpha-crystallin are positive. The Flory theta temperature was found to be 271 K.     

Studies on zinc- and cadmium-bound proteins in bovine kidneys by biochemical and neutron activation techniques

Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd-protein complexes were observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results showed the presence of at least four Zn-binding proteins with mol wt>300,000, 260,000, 89,000, and 27,000 and at least three Cd-binding proteins of mol wt>300,000, 32,000, and 13,000.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Developments in tomographic methods for biological trace element research

Neutron-induced γ-ray emission tomography for quantitative determination of the concentration and distribution of elements in a selected plane through a biological specimen is briefly explained and applied by way of illustration to the analysis of gallstones. A system capable of carrying out studies of the binding site of⁷⁵Se in different matrices using time differential perturbed angular correlation spectroscopy is also briefly described. Developments in the detector technology of positron emission tomography have allowed small-diameter imaging devices to be built for in vivo preclinical evaluation of new tracers in small animals and are discussed in the context of a proposed experiment combining the techniques mentioned above.