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A new mass spectrometric assay of N8-acetylspermidine deacetylase and partial purification of the enzyme

Author

YongYuan Zhao, University of the Pacific

Date of Award

2007

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Chemistry

First Advisor

Patrick R. Jones

First Committee Member

James W. Blankenship

Second Committee Member

Jianhua Ren

Abstract

A new enzyme activity assay has been developed for the target N⁸-acetylspermidine deacetylase, a not-well-studied but essential enzyme in the polyamine interconversion and reutilization pathway.

The enzyme assay, based on mass spectrometric detection of a specific reaction product following sample introduction by flow injection, was shown to have a sensitivity of smaller than 1 micromolar and typical RSD of 3-10%. The linear range for analyte was from 1 μM to 100 μM, with R² > 0.992. The new assay avoids the use of radio labels. Sample preparation is straightforward, and high specificity is provided by the selected reaction monitoring, SRM, using a triple quadrupole mass spectrometer.

Acetylputrescine was used for the first time as the substrate for the assay of N⁸-acetylspermidine deacetylase in lieu of N⁸ -acetylspermidine. The crude enzyme extracted from rat liver had an apparent K_m value of 80.6 μM for acetylputrescine and a V_max of 1.1 nmol mg^-1 min^-1. Enzyme extracted from frozen rat liver was compared with that from fresh rat liver. Frozen rat liver extraction had similar kinetics parameters with the fresh preparation and had a specific activity of 0.8 nmol mg^-1 min^-1.

N⁸-acetylspermidine deacetylase was partially purified by protein precipitation and gell filtration chromatography. Affinity chromatography was tentatively applied for further isolation of the enzyme, but was not yet successful.

Pages

108

Recommended Citation

Zhao, YongYuan. (2007). A new mass spectrometric assay of N8-acetylspermidine deacetylase and partial purification of the enzyme. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/683