I am doing Beta-Glucosidase assay from a hot spring archaea. The assay conditions include Sodium citrate buffer, pH 5.0, temperature at 70C and the substrates are 2-Nitrophenylglycoside and 4-Nitrophenylglycoside. How can I calculate the Molar extinction coefficient of 2-Nitrophenol and 4-Nitrophenol for the above mentioned assay conditions?
1. Prepare separately aqueous solutions of 2-Nitrophenol and 4-Nitrophenol. These are to be used as stock solutions.
2. From the above stock solutions, prepare five different concentrations of each one (say, 0.0001M, 0.00025M, 0.0005M, 0.00075M, and 0.001M).
3. In the spectrophotometer UV-Vis one can select light sources (UV and visible), choose the slit width,scan speed and transmittance or absorbance display, wavelength range of interest, etc.
4. Take two clean and dry glass or quartz cuvettes with a given path length (witdth) (say, 1 cm).
5. Fillup one cuvette with blank solution (distilled water) and the other cuvette with a prepared solution with lowest concentration.
6. Place the blank in the reference holder and the sample in the sample holder.
7. Run the scan in the spectrophotometer.
8. Record the wavelength at which occurs the maximum absorbance.
8. Similarly spectral runs are done for all the other samples starting from the lowest concentrations to next higher concentrations. Keep using the same max wavelenght and recording its absorbance. Every time one should rinse the cuvette using a small portion ofthe solution from the solution that will be measured in next.
9. Make a table containing these data (Max Absorbance vs Concentration).
10. Construct a calibration plot by plotting Absorbance versus Concentration for such a wavelength.
11. The slope of the calibration plot is the Molar Extinction Coefficient of your substance at your analysis conditions.
(Adapted from http://iiith.vlab.co.in/?sub=19&brch=206&sim=567&cnt=973)
A well, simple summarized procedure is posted here too:
http://answers.yahoo.com/question/index?qid=20090129062225AA1iegu
Best regards
Ruben de Regil
The slope of a standard curve of 2-nitrophenol or 4-nitrophenol vs absorbance at the pH of the assay is the molar extinction coefficient. Watch your units!
Ismael
1. Prepare separately aqueous solutions of 2-Nitrophenol and 4-Nitrophenol. These are to be used as stock solutions.
2. From the above stock solutions, prepare five different concentrations of each one (say, 0.0001M, 0.00025M, 0.0005M, 0.00075M, and 0.001M).
3. In the spectrophotometer UV-Vis one can select light sources (UV and visible), choose the slit width,scan speed and transmittance or absorbance display, wavelength range of interest, etc.
4. Take two clean and dry glass or quartz cuvettes with a given path length (witdth) (say, 1 cm).
5. Fillup one cuvette with blank solution (distilled water) and the other cuvette with a prepared solution with lowest concentration.
6. Place the blank in the reference holder and the sample in the sample holder.
7. Run the scan in the spectrophotometer.
8. Record the wavelength at which occurs the maximum absorbance.
8. Similarly spectral runs are done for all the other samples starting from the lowest concentrations to next higher concentrations. Keep using the same max wavelenght and recording its absorbance. Every time one should rinse the cuvette using a small portion ofthe solution from the solution that will be measured in next.
9. Make a table containing these data (Max Absorbance vs Concentration).
10. Construct a calibration plot by plotting Absorbance versus Concentration for such a wavelength.
11. The slope of the calibration plot is the Molar Extinction Coefficient of your substance at your analysis conditions.
(Adapted from http://iiith.vlab.co.in/?sub=19&brch=206&sim=567&cnt=973)
A well, simple summarized procedure is posted here too:
http://answers.yahoo.com/question/index?qid=20090129062225AA1iegu
Best regards
Ruben de Regil
I don't know if you can measure at 70C and what effect that will have on the adsorbance. At 30 C, 4NP has a very low absorbance at around 400 nm and at lower wavelengths overlaps with the 4NP-Glc absorbance. Anyway, try the above mentioned method to see the extinction coefficient if you want to do a continuous assay, but you may need to stop the reaction with 2 M sodium carbonate to bring the pH up to around 8 or 9 (significantly above the pKa of the product) and measure. You should still determine the extinctioin coefficient yourself with the standard in your buffer system.
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