Masters Theses

Date of Award

12-1981

Degree Type

Thesis

Degree Name

Master of Science

Major Professor

L. Bruce Jacobson

Committee Members

Nelwyn T. Christie, Keith Owenby

Abstract

A rapid, fluorometric assay for sepiapterin production has been developed. Excitation and emission wavelengths which minimized the fluorescent contributions of H2NTP, NADPH, and pigments in the enzyme extract were defined. Further reductions in background fluorescence were obtained by chromatographing the enzyme extract over G25 Sephadex to reduce the amount of pteridines present and by stopping the reaction with EDTA. Fluorescence due to sepiapterin was defined as the difference in relative fluorescence intensity of sample mixture with and without EDTA added prior to incubation. Control reaction mixtures showed increased fluorescence intensity upon incubation; HPLC analysis of these mixtures showed that they did not contain sepiapterin. The fluorometric assay is limited by two photophenomena. Quenching, which was significant at relative fluorescent intensities of 250 or more, could be eliminated by appropriate dilution of the intensely fluorescent samples. Sepiapterin fluorescence was temporarily diminished by prolonged exposure to the excition wavelength or room light. A maximum fluorescent response could be ensured by holding samples in the dark for three minutes before measuring their relative fluorescence intensities.

Samples of known sepiapterin concentration were used to establish a ratio of fluorescence intensity units per µM sepiapterin. This conversion factor was used to determine relative sepiapterin concentrations in sample reaction mixtures. The validity of the fluorometric assay was tested by comparing sepiapterin concentrations measured fluorometrically and by HPLC. There was excellent correlation between the two methods. The fluorometric assay was used to measure the relative amount of sepiapterin synthase activity in Drosophila containing the purple mutation plus zero, one, or two copies of a "suppressor of purple" mutation. Activity was not increased in flies with one copy of the suppressor mutant, however in flies with two copies, the level of activity was restored to 47% that of wild type. GTP cyclohydrolase I, the enzyme which produces the starting material for the production of sepiapterin, has been isolated using GTP Sepharose affinity chromatography as described by Yim and Brown (1976). Experiments were performed to determine the reasons for poor GTP binding and low yields in earlier purifications. GTP binding to the activated Sepharose was markedly improved when the column material was stirred in a rotary stirring device rather than with a magnetic stirrer. Overall yield was somewhat improved following this change. Increasing the amount of NaI04used to activate GTP for attachment to the Sepharose did not have a deleterious effect on GTP binding. Two published methods for the preparation of GTP Sepharose (Lamed et al., 1973; Jackson et al., 1973) were compared with the standard laboratory method. No differences in the amount of GTP binding or yield were observed. The rate of application of enzyme to the GTP Sepharose column was found to make no difference in the amount of enzyme bound to the column or in the amount of activity recovered.

Sepiapterin synthase has been purified by chromatography on Cibacron blue Sepharose (Krivi and Brown, 1979). Specific binding is presumed to occur between the blue dye and the NADPH-requiring activity of the enzyme. Seven millimolar NADH was found to be ineffective in eluting activity from the column. Elution with buffer containing 1 M NaCl could remove activity from the column even after previous elution with NADPH-containing buffer. The purification described by Krivi and Brown (1979) was repeated without success.

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