The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media.

synopsis This paper describes the first experimental application of fluorescence correlation spectroscopy, a new method for determining chemical kinetic constants and diffusion coefficients. These quantities are measured by observing the time behavior of the tiny concentration fluctuations which occur spontaneously in the reaction system even when it is in equilibrium. The equilibrium of the system is not disturbed during the experiment. The diffusion coefficients and chemical rate constants which determine the average time behavior of these spontaneous fluctuations are the same as those sought by more conventional methods including temperature-jump or other perturbation tecliniques. The experiment consists essentially in measuring the variation with time of the number of molecules of specified reactants in a defined open volume of solution. The concentration of a reactant is measured by its fluorescence; the sample volume is defined by a focused laser beam which excites the fluorescence. The fluorescent emission fluctuates in proportion with the changes in the number of fluorescent molecules as they diffuse into and out of the sample volume and as they are created or eliminated by the chemical reactions. The number of these reactant molecules must be small to permit detection of the concentration fluctuations. Hence the sample volume is small (10-8 rnl) and the concentration of the solutes is low (-10-9 M). We have applied this technique to the study of two prototype systems: the simple example of pure diffusion of a single fluorescent species, rhodamine 6G, and the more interesting but more challenging example of the reaction of macromolecular DNA with the drug ethidium bromide to form a fluorescent complex. The increase of the fluorescence of the ethidium bromide upon formation of the complex permits the observation of the decay of concentration fluctuations via the chemical reaction and consequently the determination of chemical rate constants.

Fluorescence Correlation Spectroscopy. II. An Experimental Realization

Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a very powerful tool for studying the diffusion behaviour of macromolecules in solution. The diffusion coefficient, and hence the hydrodynamic radii calculated from it, depends on the size and shape of macromolecules. In this review, we provide evidence of the usefulness of DLS to study the homogeneity of proteins, nucleic acids, and complexes of protein–protein or protein–nucleic acid preparations, as well as to study protein–small molecule interactions. Further, we provide examples of DLS’s application both as a complementary method to analytical ultracentrifugation studies and as a screening tool to validate solution scattering models using determined hydrodynamic radii.

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Dynamic light scattering: a practical guide and applications in biomedical sciences.

On the function and mechanism of action of peroxidases

Challenges in the development of high protein concentration formulations

The Brownian motion of a single sample of high-molecular-weight poly(L-lysine) [(Lys)n, n = 955] has been studied by dynamic light scattering over a wide range of NaBr concentrations and at three different polyion concentrations. A substantial decrease in scattered intensity is associated with the transition from the ordinary phase to the low-salt extraordinary phase. At the salt concentration where the transition takes place the relaxations are non-exponential and appear to exhibit at all angles a rapid relaxation (τ ≅ 10 μsec) that is presumed to be a manifestation of the kinetics of the transition process. The K2 dependence of the slow relaxation rates in the extraordinary phase has been confirmed within the experimental error. The extrapolated infinite-dilution values of the diffusion coefficients in the ordinary phase are observed to decline precipitously below 10−2M salt to astonishingly small values, indicating a dramatic rise in the friction factors of the isolated polyions. An extensive discussion of these findings in relation to the theory employed here and to existing data in the literature is also given.

Brownian motion of highly charged poly(L-lysine). Effects of salt and polyion concentration

Role of orientation constraints and rotational diffusion in bimolecular solution kinetics

Torison dynamics and depolarization of fluorescence of linear macromolecules. II. Fluorescence polarization anisotropy measurements on a clean viral phi 29 DNA.

Of the physical techniques available for the study of biopolymers and other macromolecular and colloidal systems, dynamic light scattering is, perhaps, the most fascinating. The very idea of extracting useful dynamical and structural information from random fluctuations in the intensity of light scattered from otherwise unperturbed equilibrium solutions1-3 seems, at first glance, almost too good to be true. Indeed, for fragile preparations that are prone to suffer undesirable fates at the slightest change in solution conditions, dynamic light scattering may be the perfect technique. It is nondestructive, relatively rapid, and typically requires only modest amounts of material. In routine applications, even on unfamiliar samples, the dust-removal procedure (either gravity flow Millipore ® filtration or low-speed centrifugation) requires 10 to 20 min, and determination of the translational diffusion coefficient to an accuracy of a few percent, or less, requires 5 min. Verification that the dynamical...

Dynamic light scattering of biopolymers and biocolloids.

The structures of viral φ29 DNA condensed by triamines, principally spermidine, in 10−3M NaCl were investigated by static and dynamic light scattering and electron microscopy. All of the results for DNA condensed in 30 μM spermidine at neutral pH are quantitatively consistent with a toroidal structure with a mean outer diameter of 1850 A. At pH 10.2, however, condensed structures of a completely different size and shape are observed for the first time. These structures are also more irregular in shape and more polydisperse than those at neutral pH. This conformational change is believed to result from a change in the mode of spermidine binding that is coupled to, or associated with, the (premature) titration of protons on the base-ring nitrogens of guanine and thymine. Besides spermidine, certain homologs of spermidine, in which the butyl moiety of spermidine was replaced by longer pentyl through octyl moieties, were also studied. Though all of the triamines condensed the DNA at 30 μM, aggregation became a more prevalent occurrence as the length of the end chain increased. This suggests that crosslinking may play an important role in the condensation process. Finally, these aggregates are dissociated to a considerable extent at pH 10.2, and the resulting compact structures appear to be quite similar, independent of the triamine used to condense the DNA. The observed partial breakdown of aggregates is also consistent with the hypothesis of a change in mode of triamine binding at pH 10.2.

J. Michael Schurr

Papers

Brownian motion of highly charged poly(L-lysine). Effects of salt and polyion concentration

Role of orientation constraints and rotational diffusion in bimolecular solution kinetics

Torison dynamics and depolarization of fluorescence of linear macromolecules. II. Fluorescence polarization anisotropy measurements on a clean viral phi 29 DNA.

Dynamic light scattering of biopolymers and biocolloids.

Structure of viral phi 29 DNA condensed by simple triamines: a light-scattering and electron-microscopy study.