A semiempirical theory of the optical activity of saccharides

A semiempirical theory of optical activity of saccharides is developed that (a) goes beyond previous empirical treatments, (b) yields calculated NaD rotations which correlate well with experimental data, and (c) accounts for a characteristic c.d. band observed for polysaccharides in the vacuum u.v. spectrum.     

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.     

STUDIES ON THE AMOUNT OF LIGHT EMITTED BY MIXTURES OF CYPRIDINA LUCIFERIN AND LUCIFERASE

1. A photometric method was devised for measuring the intensities of light emitted per cc. of hiciferin solution and calculating the amount of light emitted per gm. of dried Cypridina powder. A total of 128 runs was made and the data are incorporated in this report. 2. The maximum amount of light emitted from 1 gm. of powder under the experimental conditions was 0.655 lumens. Different samples of powder vary greatly in amount of light production. 3. When the concentration of substrate is doubled, nearly twice as much light is emitted, or an average ratio 2C/C of 1.86. Calculations of total light emissions per gm. of powder at different concentrations indicate that slightly more light is produced from the smaller concentrations. The maximum amount of light was produced by the solutions made with neutral sea water and averaged 0.445 lumens. The least light was obtained from solutions in distilled water saturated with hydrogen. The technique allows too rapid spontaneous oxidation prior to the saturation with hydrogen. The maximum amount of light from such experiments was only 0.077 lumens. Acid sea water solutions subsequently neutralized gave an average maximum of 0.386 lumens per gm. of powder per second. 4. When the concentration of enzyme is doubled, approximately the same amount of light is produced by both concentrations, although the stronger concentrations are slightly less effective than weaker ones. This undoubtedly is due to the colloidal nature of the enzyme and is a function of surface rather than of mass. In dilute solutions greater dispersion probably allows for greater adsorption to the surface of the enzyme. The average maximum amount of light produced in the series of enzyme experiments is of the magnitude 0.56 lumens per gm. of powder.