Date of Award

Summer 1998

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Biochemistry

Committee Director

Mark S. Elliot

Committee Member

Rana C. Morris

Committee Member

Roy L. Williams

Committee Member

James H. Yuan

Call Number for Print

Special Collections LD4331.B43 H69

Abstract

Reverse transcriptase (RT) is an RNA-directed DNA polymerase isolated from human immunodeficiency virus (HIV) and other retroviruses. It has been the primary target for anti-HIV research since the discovery of its vital role in the retroviral life-cycle. The conventional method for carrying out kinetic characterization and inhibition studies of reverse transcriptase involves the use of radioactive materials. Although this method is effective, it is both time consuming, due to a necessary repeated washing step, and hazardous to the worker and environment. Furthermore, strict laws limit the use of radioactive materials to licensed workers and laboratories. In response to these concerns, we have developed a novel method for carrying out the kinetic analysis of reverse transcriptase through the use of the fluorescent dye 4',6-diamidino-2- phenylindole (DAPI). This method does not require repeated washings or special licensing and poses minimal health risks to the worker. We performed steady state kinetic analysis of two reverse transcriptase enzymes, HIV-1 RT and MMLV RT, by the fluorometric technique and the conventional radiochemical method. Binding constants were calculated in both methods for comparison. In addition, experimental HIV-1 RT values were compared to literature values. In all cases, the results were in relative agreement, verifying the fluorometric method as an alternative to the conventional radiochemical method. This method may be applied to future kinetic and inhibition studies of reverse transcriptase.

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DOI

10.25777/9gk1-qx82

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