published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

A rotating ring-disk electrode (RRDE) has been used to study heterogeneous catalytic reactions involving an immobilized enzyme. Glucose oxidase (E.C. 1.1.3.4.) has been immobilized by covalent attachment to a variety of disk electrode supports resulting in the rotating ring-disk enzyme electrode (RRDEE). Covalent attachment to graphitic oxide, platinum and carbon paste has been achieved using the bifunctional reagents glutaraldehyde or 1-ethyl-3(3-dimethyl amino-propyl)-carbodiimide. By varying the electrode rotation speed, the effects of external substrate mass transport on the rate of enzymatic catalysis have been investigated. Extremely small diffusion boundary layers (ca. 10-25 μm) at the disk catalytic support, under conditions of well defined and reproducible hydrodynamics, facilitate this investigation. The rate enzymatic catalysis is evaluated by spectrophotometrically monitoring the formation of the product H₂O₂ in the bulk solution. Peroxide may also be conveniently monitored amperometrically at the concentric platinum ring giving a steady-state response proportional to the substrate concentration. It is assumed that the enzyme obeys Michaelis-Menten kinetics. The intrinsic value of the heterogeneous apparent Michaelis-Menten constant (K'(m)) has been determined from Lineweaver-Burk plots for both methods of product detection. Catalysis limited rates are observed only when the electrode rotation speed is high (ω ≥ 1600 rpm) as established from linear Lineweaver-Burk plots and by calculating characteristic dimensionless parameters relating the ratio of the rate of catalysis to substrate mass transfer (e.g. the Damkoehler number and reaction velocity parameter). A careful characterization of the "immobilized enzyme layer" was necessary to evaluate the RRDEE as a viable model for these investigations. The specific activity of the immobilized enzyme was measured and related to the disk supports, immobilization procedures, and subsequent enzyme loading. Specific activities range from Whereas the effects of external mass transfer resistances may be eliminated at high rotation speeds, any internal or intra-enzyme layer resistances may not. Using chronoamperometric techniques for several disk electroactive species, it was shown that the the effect diffusion coefficients of small molecules within the enzyme layer are approximately 25 - 50% of the bulk solution value. K'(m) determined from the ring amperometric measurements was found to be slightly larger than values determined from the bulk solution detection of product and was attributed to the interenzyme diffusion of product. This, however, was shown not to interfere with product detection under conditions of catalysis limited rates.

Ornithine decarboxylase has been purified to apparent homogeneity from thioacetamide-stimulated calf liver. The purification process, which has been developed to circumvent the lability of the enzyme, employs ion exchange chromatography, gel filtration, hydroxylapatite chromatography, non-denaturing gel electrophoresis, and sulfhydryl affinity chromatography. The enzyme is purified 71,500-fold to a final specific activity of 286,000 pmol/min/mg protein. Non-denaturing gel electrophoresis indicates a single protein present in the final preparation. The enzyme has a Stokes radius of 3.14 nm as indicated by gel filtration and a monomeric molecular weight of 52,000 daltons as indicated by denaturing gel electrophoresis. The K(m) values for ornithine and pyridoxal phosphate are 0.16 mM and 2.5 μM, respectively. Putrescine inhibits the enzyme (Kᵢ 10mM). The existence of three ionic forms of ornithine decarboxylase is suggested by fractionation of the preparation by gradient sievorptive chromatography. Mammalian ornithine decarboxylase is apparently a metalloenzyme. A variety of structurally distinct metal chelators inhibit the enzyme. A non-chelating analog of the most potent chelator, 1,10-phenanthroline, is without effect. The order of efficacy of the chelators suggests the involvement of a metal from the transition series. Incubation of the enzyme with charcoal or Cibacron Blue-Agarose results in a loss of catalytic activity suggesting that the ornithine decarboxylase may also contain a bound nucleotide.

No description available.

No description available.

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No description available.