Agarose conformation

FIGURE 7.31 The favored conformation of agarose in water is a double helix with a threefold screw axis. 

Small-angle X-ray scattering (SAXS), circular dichroism (CD), and UV spectroscopy at different temperatures were used to investigate the nature of calf-thymus DNA in aqueous solution, in the presence of [Me Sn] " (n = 1-3) species. The results demonstrate that the [MeSn(IV)] moiety does not influence the structure and conformation of the DNA double helix, and does not degrade DNA, as indicated by agarose gel electrophoresis. Inter alia, the radii of gyration, Rg, of the cross section of native calf-thymus DNA, determined by SAXS in aqueous solution in the presence of [Me Sn] " (n = 1-3) species are constant and independent of the nature and concentration of the [Me Sn] species. 

It is noteworthy that most of the chemical shift values for all three polymers may be closely approximated ( ) by calculations based on data for monomeric reference compounds. These findings illustrate, therefore, the general validity of studies on low molecular weight model compounds for einalysis of spectra of carbohydrate polymers. Many examples of equally satisfactory comparisons of this kind are to be found in studies on other polysaccharides (11,23). These polymers include glucans (l6), mannans (2k, 2 ), limit dextrins (26), lichenin (2j), agarose (28) and various polysaccharides of fungal and microbial orgins (e.g., 7,8,29-31). Observed departures from expectation have been attributed to specific conformational influences ( 8). 

Currently, the best way of preserving the native conformation under the conditions of elution chromatography is hydrophobic interaction chromatography with minimized adsorption forces. These can be adjusted by selecting the appropriate stationary phase, e.g., from a series of alkylated agarose gels with various alkyl chain lengths. 

Dea, I. C. M., McKinnon, A. A., and Rees, D. A. (1972). Tertiary and quaternary structure in aqueous polysaccharide systems which model cell wall cohesion reversible changes in conformation and association of agarose, carrageenan and galactomannans. J. Mol. Biol. 68 153-172. 

Sela, L, Fluorescence of nucleic acids with ethidium bromide an indication of the configurative state of nucleic acids, Biochim. Biophys. Acta 190, 216-219, 1969 Le Pecq, J.B., Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. Methods Biochem. Anal. 20, 41-86, 1971 Borst, P., Ethidium DNA agarose gel electrophoresis how it started, lUBMB Life 57, 145-141, 2005. 

Braudo, E. E., Muratalieva, 1. R., Plashchina, 1. G., and Tolstoguzov, V. B. 1991. Correlation between the temperatures of formation breakdown of the gel network and conformational transitions of agarose macromolecules. Carbohydr. Polym. 15 317-321. 

Experiments performed in a very similar way also showed dynamic disorder for the enzyme a-chymotrypsin [16], a-Chymotrypsin is an endopeptidase acting on water-soluble polypeptides. The substrate (suc-AAPF)2-rhodamine 110 was designed to interact optimally with the binding site of the enzyme. It consists of a rhodamine 110 core that is derivatized with a succiny-lated AlaAlaProPhe peptide sequence, known to bind very specifically at the enzyme s active site (Fig. 25.2a). To further avoid potential artifacts, the enzyme was immobilized by entrapment in an agarose matrix, which restricts enzyme diffusion while still allowing free rotation and conformational dynamics of the enzyme as well as the diffusion of the substrate. 

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