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Dihydrofolate reductase is a ubiquitous enzyme present in all eukaryotic and prokaryotic cells, playing a key role in thymidine synthesis. It catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8- tetrahydrofolate (THF), utilizing NADPH as cofactor.This reaction is an essential step in the biosynthesis of nucleotidic bases of DNA.1-3 Blockage of the DHFR enzyme causes cell death as a result of DNA synthesis inhibition. For this reason, DHFR is considered an excellent target for designing antitubercular drugs.
All the synthesized derivatives of the substituted benzthiazole/benzimidazoles were evaluated for DHFR inhibitory activity using the Dihydrofolate Reductase Assay Kit . The Kit is designed for the detection of DHFR inhibitory activity and for screening DHFR inhibitors. It provides all the reagents required (including a purified enzyme) for the efficient detection of DHFR activity and inhibition in cell lysates, tissue homogenates, or column fractions of purified enzyme
Dihydrofolate Reductase (DHFR)- 0.1 unit
Assay Buffer 10X for DHFR -30 ml
Dihydrofolic acid (DHFR substrate)-3 X 10 mg
Methotrexate (DHFR inhibitor) -2 X 10 mg
NADPH (Nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt) -25mg
Equipment required :
· Temperature controlled UV/visible spectrophotometer.
· 1 ml Quartz cuvette
· Ultrapure water:
Principle of assay for DHFR activity
The assay is based on the ability of Dihydrofolate reductase to catalyze the reversible NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid.
Dihydrofolic acid+NADPH+H+ Tetrahydrofolic acid+NADP+
At pH 7.5, the equilibrium of the reaction lies relatively far to the right, and the reaction goes essentially to completion in the forward direction. The reaction progress is monitored by the decrease in absorbance at 340 nm.
Preparation of reagents:
1. Dihydrofolic acid (substrate)
A 10 mM stock solution was prepared by maintaining pH 7.5 by the addition of 2.2 ml assay Buffer 10X to the dihydrofolic acid bottle (i.e., add 2.2 ml Assay Buffer 10X to 10 mg powder), and mixed well. Aliquot the 10 mM dihydrofolic acid stock solution was taken and stored at -20 °C. The remaining unused solutions was discarded on the same day.
2. 10 mM NADPH stock solution
A 10 ml suspension buffer was prepared by adding 0.2 ml Assay Buffer 10X to 9.8 ml water. 3 ml of the suspension buffer was added to the NADPH bottle. Mixed well and the solution was stored at -20 °C.
3. Standard- (Methotrexate-inhibitor) stock solution
A 10 mM stock solution was prepared by adding 2.2 ml of Assay Buffer 10X to the bottle. Mixed well and stored it at -20 °C.
4. Assay Buffer 10X
Assay Buffer 10X was diluted for DHFR ten fold in deionized water (i.e. add 5 ml Assay Buffer 10X for DHFR to 45 ml water) kept at room temperature.
All the synthesized compounds were prepared at a final concentration of 2 mg/ml reaction mixture.
The amount of DHFR supplied in the kit is approximately 0.1 units. The volume of the enzyme (in ml) to be used was calculated using the formula, Volume (ml) = 1.5 X 10-3 X 1000/(units/mg protein) X (mg protein/ml) . According to the lot specific data, the volume of enzyme to be used usually varies between 10-30 ml
Spectrophotometer was set at 340 nm and 22 °C. Assay Buffer 10X was added to the test microcentrifuge tube and to which DHFR enzyme or the sample was also added ,mixed well. The synthesized compounds BTZ(1-13) and BIM( 1-12 )was also added to the appropriate tube, mixed well to which 6 µl of NADPH solution and 5 ml of dihydrofolic acid were added .The contents of the tube were transferred to a 1 ml quartz cuvette , mixed and immediately inserted the cuvette into the spectrophotometer. The kinetics program was started immediately. The decrease in optical density (DOD) obtained were recorded as absorbance at 340 nm . Results were tabulated and compared with standard methotrexate drug.
The absorbance at 340 nm will decrease (due to decrease in NADPH concentration). 10-20 ml of the supplied DHFR enzyme will usually give a linear slope during the 2.5 minutes of the detection.
The decrease in DOD obtained during 2.5 min.as DOD/min was measured.
The activity was caluculated using the formula:Units/mg
P = (DOD/min.sample - (DOD/min.blank) X d/12.3 X V X mg P/ml
DOD/minblank: DOD/min. for the blank, from the spectrophotometer readings
DOD/minsample: DOD/min. for the reaction, from the spectrophotometer readings
12.3: extinction coefficient (e, mM-1cm-) for the DHFR reaction at 340 nm.
V: Enzyme volume in ml (the volume of enzyme used in the assay)
d: The dilution factor of the enzyme sample.
mg P/ml: enzyme concentration of the original sample before dilution.
Units/mg P: - Specific activity in mmol/min/mg protein