Dihydrofolatereductase (DHFR) is an important enzyme in the formation of DNA (Goodsell, 2002). DHFR controls the amount of tetrahydrofolate in the cells, which in turn is an important cofactor for purine and thymidylate synthesis and ultimately in the growth and proliferation of the cell (Davidson &Sittman, 1999). The enzyme is common to many species on earth. Human and bacterial DHFR have a relatively low rate of primary sequence agreement at 26%. However, they both retain a common general structure.The key role that the enzyme plays in biology as well as the variations that the enzyme displays makes it an important target of antibacterial and anticancer drugs (Jarabak&Bachur, 1971).
The physiologic role of DHFR is the reduction of Folic Acid to 7,8 dihydrofolate, which is produced in a reaction of thymidylate synthase and autooxidation by tetrahydrofolate. Folic acid alleviates symptoms of folate deficiency anemia and is widely found as a pro-vitamin (Bailey &Ayling, 2009). However, it has been widely reported that increased folic acid loads may exacerbate and accelerate other cancers. Furthermore, high levels of folic acid have been linked to immune deficiencies and a decreased natural killer cell activity in older women. These disease processes are hypothesized to be related to incomplete metabolism of folic acid in the tissues (Bailey &Ayling, 2009). Thus, it is important to characterize the enzyme that degrades folic acid. DHFR activity varies widely throughout the tissues that utilize the enzyme. Measurement of the enzyme’s activity is useful because it may help in the detection of cancers by aiding to locate areas that are poorly utilizing the DFHR enzyme (Bailey &Ayling, 2009).
The purification of DHFR is multistep and partially dependent on the tissues it’s extracted from. When extracted from human placentas the steps are: homogenization, ammonium sulfate precipitation, CM-Cellulose chromatography, first Sephadex g-100 gel filtration, second spehadex g-100 gel filtration, hydroxylapatite chromatography, CM-Sephadex chromatography, and hydroxylapatite concentration (Jarabak&Bachur, 1971). During the entire process, pH and temperature must be carefully controlled so as not to denature the proteins that are being collected. Various buffers are used to help control the pH levels of the sample (Jarabak&Bachur, 1971). In the purification experiments conducted by Jarabak and Bachur, they showed the small differences that exist in DHFR enzyme structure that exists between the DHFR in human placenta and rat, mouse, and chicken liver variants of the enzyme. The basic procedure for purifying mammalian DHFR is the same throughout the various tissues and species.
DHFR enzyme activity can be assessed by performing an enzyme assay; more specifically a spectrophotometric continuous assay (Alam, 1986). All type of enzyme assays measure the consumption of substrate in a biochemical process over time or it can measure the amount of product produced over time. In spectrophotometric continuous assay, the reaction that the enzyme catalyzes goes on, one can measure the amount of light that the assay solution absorbs. UV light is used most often because the coenzymes NADH and NADPH absorb UV light in their reduced states but not in the oxidized forms (Alam, 1986).
DHFR is an important enzyme in mammalian cells. Besides the regulation of our own DNA, we are also able to use this enzyme and specifically the inhibition of this enzyme in medical practice in infection with bacteria and in cancer; therefore understanding of it activity and is important for the future treatment of these conditions.
References
Goodsell, D. (2002) DihydrofolateFolateReductase. Protein Data Bank (PDB) http://dx.doi.org/10.2210/rcsb_pdb/mom_2002_10
Wellman, S., &Sittman, D. (1999). Nucleotide Metabolism. In V.L. Davidson & D.B. Sittman (Eds.).NMS Biochemistry (403-424).Philadeliphia: Lippincott Williams & Wilkins.
Bailey, S.W., and Ayling, J.E. (2009). The extremely slow and variable activity of
dihydrofolatereductase in human liver and its implications for high folic acid Intake. PNAS, 106(35), 15424-15429.www.pnas.org_cgi_doi_10.1073_pnas.0902072106.
Jarabak, J., and Bachur, N.R. (1971). A Soluble DihydrofolateReductase from Human
Placenta: Purification and properties. Archives of Biochemistry and Biophysics, 142, 417-425.
Alam, A. (1986). Large scale production and C]characterisations of dihydrofolatereductase from a methotrexate – resistant human lymphoid cell line. Journal of Bioscience, 10(1)37-47
