In vitro studies of Thiopurine S-Methyltransferase: Ligand binding interactions and development of a new enzymatic activity assay for TPMTwt, TPMT*6 and TPMT*8
Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity.
In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX.
Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.
Place, publisher, year, edition, pages
2015. , 34 p.
Thiopurine S-Methyltransferase, 6-mercaptourine, methotrexate, isothermal titration calorimetry, fluorescence spectroscopy
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-119652OAI: oai:DiVA.org:liu-119652DiVA: diva2:825493
Subject / course