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Assays were performed on individual Escherichia coli β-galactosidase molecules at 2 different concentrations of the substrate DDAO-β-d-galactoside using a free zone capillary electrophoresis-based protocol with an in-laboratory-constructed instrument utilizing laser-induced fluorescence detection. In a typical run, 2 enzyme molecules were injected into the capillary. They were separated from each other by a brief period of electrophoresis and incubated on the capillary in the presence of the substrate. They were then mobilized on the capillary into a zone of substrate at a different concentration, re-incubated, and the product peaks mobilized past the detector . The relative change in activity as the concentration was increased differed between molecules, suggesting differences in Km. In a different experiment, the capillary was filled with on average 13 enzyme molecules per run, incubated, and the activities of the individual molecules determined. The shapes of the distribution curves of single molecule activities obtained at different concentrations of the substrate resorufin-β-d-galactoside were indistinguishable, suggesting a homogeneous Km. To explain why individual enzyme molecules behaved as if they were heterogeneous with respect to Km but the population behaved as if it were homogeneous, theoretical Michaelis-Menten curves were constructed. The curves for populations with heterogeneous Km values were found to be indistinguishable from that of a homogeneous population. Des tests ont été réalisés sur des molécules individuelles de β-galactosidase d’E. coli mises en présence de deux concentrations du substrat DDAO-β-d-galactoside en utilisant un protocole basé sur l’électrophorèse capillaire de zone libre à l’aide d’un instrument de détection de fluorescence induite par laser construit dans le laboratoire. Lors d’un test typique, deux molécules d’enzyme ont été injectées dans le capillaire. Elles ont été séparées l’une de l’autre par une brève période d’électrophorèse et ont été incubées dans le capillaire en présence de substrat. Elles ont ensuite été mobilisées dans le capillaire dans une zone où le substrat se trouvait à concentration différente et ré-incubées, puis les pics de produits ont été mobilisés après le détecteur. Le changement relatif d’activité en fonction de l’augmentation de la concentration différait entre les molécules, suggérant des différences de Km. Lors d’une autre expérience, le capillaire a été rempli avec une moyenne de 13 molécules d’enzyme par essai, incubé et l’activité des molécules individuelles a été déterminée. La forme des courbes de distribution des activités des molécules individuelles obtenues à différentes concentrations du substrat résorufine-β-d-galactoside était la même, suggérant un Km­ homogène. Afin d’expliquer pourquoi les molécules individuelles d’enzyme se comportaient comme si elles étaient hétérogènes en termes de Km mais que la population se comportait comme si elles étaient homogènes, des courbes théoriques de Michaelis-Menten ont été construites. Les courbes des populations de molécules à valeurs de Km hétérogènes étaient indiscernables de celles de la population homogène. [ABSTRACT FROM AUTHOR]

Chitinases are enzymes that serve critical roles in fungal growth and development, in resistance of plants to fungal pathogens, and in parasitism of insects by entomopathogenic fungi. The term "chitinase" is used for 3 enzymatic activities: N-acetylglucosaminidases, which sequentially release N-acetylglucosamine residues from the chitin polymer; chitobiosidases, which release disaccharides; and endochitinases, which cleave within the polymer and release oligosaccharides. We describe a technique where chitinases are separated on non-denaturing polyacrylamide gels, activities are visualized and characterized with chitinase specific substrates, and specific activities are estimated by image analysis. This technique permits a rapid determination of all of the types of chitinases present within a sample as well as their activities.Key words: chitinases, electrophoresis, non-denaturing gels. [ABSTRACT FROM AUTHOR]

Taxol (paclitaxel) is an anticancer drug that interacts with microtubule proteins in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures. However, in the human lung tumor cell, the concentration of paclitaxel is highest in the nucleus. Therefore, it was of interest to examine the interaction of taxol with DNA and RNA in aqueous solution at physiological pH. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to characterize the nature of drug–DNA and drug–RNA interactions and to determine the taxol binding site, the binding constant, the sequence selectivity, the helix stability, and the biopolymer secondary structure in the taxol–polynucleotide complexes in vitro. The FTIR spectroscopic studies were conducted with taxol/polynucleotide (phosphate) ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2 with a final DNA(P) or RNA(P) concentration of 12.5 mmol/L, and capillary electrophoresis was performed after incubation of taxol with polynucleotides at ratios of 1/200 to 1/12 with a final polynucleotide concentration of 1.25 mmol/L. Taxol was shown to bind to DNA and RNA at G–C, A–T, or A–U bases and the backbone PO2 group. Two types of binding were observed for taxol–DNA with K1 = 1.3 × 104 L mol–1 and K2 = 3.5 × 103 L mol–1, whereas taxol–RNA complexes showed one type of binding with K = 1.3 × 104 L mol–1. The taxol–polynucleotide complexation is associated with a partial helix stabilization and no major alterations of B-DNA or A-RNA structure. [ABSTRACT FROM AUTHOR]

Non-equilibrium molecular dynamics (MD) of small, charged cadmium selenide nanorods have been carried out in the absence and presence of static applied electric fields. In the absence of applied fields, it was found that opposite dipolar alignment (antiferromagnetic) was achieved, along with self-assembly of the nanorods. However, in the case of induced electrophoresis in applied fields, the rods approached each other less readily, while at and above a field intensity of 0.05 V/Å, preferential alignment with the field was achieved for all rods, in contrast to the zero-field case. These results have implications for electric field-mediated control of nanorod assembly in solution, of key importance in a wide range of areas from photovoltaics to energy storage. [ABSTRACT FROM AUTHOR]