SynopsisBradley et al. [(1972) Biopolyrners 11,645-6521 used electro-optical measurements to show that methylene blue (MB), like acridine orange, in its DNA complex is oriented more or less perpendicular to the helix axis as expected if intercalated. High-precision flow linear dichroism (LD) here confirms that MB is coplanar with the DNA bases at low dyeDNA binding ratios and low ionic strengths. CD shows two origins of induced optical activity for the transition of lowest energy (polarized parallel to the long-axis of the dye molecule): a t low binding ratios (r < 0.05), a weak monomeric CD with the same shape as the absorption curve is observed, while at higher binding ratios, a strong exciton CD dominates due to interaction between pairs of MB ligands. The monomeric CD spectrum shows a remarkable dependence on ionic strength: it is negative in the absence of extra salt, but changes sign (at ca 20 mM NaCl or 0.15 mM MgC12) and becomes essentially the positive mirror-image spectrum at high ionic strengths (>300 mM Na+ or >0.4 mM Mg2+). Nondegenerate coupled-oscillator theory can explain the CD in terms of interactions of transition moments of the dye and the nearest nucleotide bases and indicates a change between two intercalation geometries: a Lerman type of mode, denoted y-, and an orthogonal mode, denoted y+. This rotation of MB in the base-pair pocket is accomplished at Na+ and Mg2+ concentrations when the phosphates are effectively screened and the result suggests a more localized bonding of Mg2+ than is expected from simple polyelectrolyte models. The exciton effect at high binding ratios, observed both in CD and in LD, can be interpreted in terms of an interaction between an intercalated and a nonintercalated MB. The geometry of this "accidental" dimer is consistent with a location of the nonintercalated MB in the minor groove, bridging the strands by the positively charged amino groups directed towards phosphate groups. The dihedral angle of the MB pairs, corresponding to a left-handed helix, is opposite to that with acridine orange and proflavine on DNA, indicating that the latter ligands bind t o DNA in a different way.
It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.
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