You run an enzyme assay designed to determine the a-glucosidase activity after 1 hour (60 min) by determining the concentration of p-nitrophenol. The results you collected are presented below. Part 1 Enzyme Dry Weight Absorbance at 580 nm : 0.290 The Required dilution factor: 30 Part 2 p-nitrophenol absorbance at 400 nm : 0.535 You now need to analyze the results and calculate the following: (Answer each question to 3 significant figures) Part 2 If the molar absorptivity of the p-nitrophenol is 7.28 mM–1·cm–1 and the path length is 1.0 cm, what is the concentration of p- nitrophenolin the sample?
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DETERMINATION OF THE λmax of p-nitrophenol: TABLE 4.6 WAVELENGTH ABSORBANCE WAVELENGTH ABSORBANCE 360 nm .332 390 nm .651 365 nm .378 395 nm .676 370 nm .439 400 nm .687 375 nm .506 405 nm .676 380 nm .562 410 nm .645 385 nm .613 415 nm .594 Calculation of concentration of p-nitrophenol added (Note: stock concentration = 0.2mM): Calculation of ε, the molar extinction coefficient: e= A/bc= .687/ ( 1cm *
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The molar extinction coefficient of p-nitrophenol is 17,500 at 400 nm and pH of 9-10. Sample: pnpp: (400 nm) 0.013 A Sample: pnpp with enzyme: (400 nm) 0.219 A What is the molar extinction coefficient, e, for para-nitrophenol, pNP, at pH 10.0? At the 15-minute point, what was the absorbance at pH 10.0? At the 15-minute point, what was the absorbance at pH 8.0? What is the molar extinction coefficient, e, for para-nitrophenol, pNP, at pH 8.0? Assume that the [pNP] at 15 minutes represents the total [pNP] at the start of the assay. What is the starting concentration of para-nitrophenyl phosphate, pNPP?
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