PubMed · 14970547
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Shawn Doonan. 2004. Making and changing buffers.. https://doi.org/10.1385/1-59259-655-x%3A91
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The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).
The retention properties of a range of N-methylated and N-des-methyl 4-substituted piperidines on polybutadiene (PBD) and polystyrene (PS) coated zirconia have been studied and compared to those of Xterra RP(18) and Genesis C(18) silica. The effect of buffer type and pH are investigated with regard to the elution order on all three stationary phases. The change in the elution order is linked to the degree of substitution of the piperidine nitrogen and appears to be independent of the rest of the structure, indicating that this moiety is most heavily involved in the separation mechanism on the zirconia phases.
The mathematical models of migration and dispersion in capillary zone electrophoresis of small molecules form a sound basis for separation strategies of complex mixtures. It turned out that the key property is the effective mobility of the sample ions. To tune resolution parameters such as pH, complexation constants and ionic strength are widely used; temperature however is not although mobilities and pK(a) values depend in a more or less degree on temperature. From the temperature dependences of pK(a) values of a number of compounds listed in the literature a general rule can be derived: for carboxylic and inorganic acids dpK(a)/dT values are very small and the pK(a) values change less than +/-0.05 units/10K. Thermodynamically speaking, these compounds exhibit dissociation enthalpies close to zero. Phenols and amines, on the other hand, have systematically larger dpK(a)/dT values of about -0.1 to -0.2 units per 10K (the results of dissociation enthalpies of 20-70 kJ/mole). Based on this classification, a distinction can be made between different situations in capillary electrophoresis: (i) selectivity changes with temperature are largely due to the temperature dependence of the pK(a) of the buffering compound in the background electrolyte, (ii) selectivity changes mainly result from the temperature dependence of the pK(a) of the sample ions, and (iii) temperature effects on the pK(a) values of both, sample and buffer play a role. This work demonstrates such effects on selectivity in capillary electrophoresis highlighting the fact that in some instances temperature can be used to fine-tune separations.