Exp_2_KEY_W12

Exp_2_KEY_W12 - Experiment 2 KEY MCB120L, W12 Overlay...

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1 Experiment 2 KEY MCB120L, W12 Overlay Spectra 1. Calculate the molar extinction coefficients, ε , for tyrosine and tryptophan at 280 nm using the overlay spectra data in Figure 2.2 on page 2-3. molar extinction coefficient, ε (M -1 cm -1 ) at 280 nm Tyr (Y) ε = (0.35 / 2.5 x 10 -4 M · 1 cm) = 1400 M -1 cm -1 Trp (W) ε = ( 1.4 / 2.5 x 10 -4 M · 1 cm) = 5600 M -1 cm -1 2. Calculate the absorption coefficients , a , for BSA, lysozyme and gelatin at 280 nm using the overlay spectra data obtained in Part A on page 2-7. absorption coefficient, a (mg/ml) -1 · (cm) -1 at 280 nm (sample values) [BSA] = 0.4 mg/ml a = (0.265 / 0.4 mg/ml · 1cm) = 0.66 (mg/ml) -1 (cm) -1 [lysozyme] = 0.4 mg/ml a = (1.010 / 0.4 mg/ml · 1cm) = 2.53 (mg/ml) -1 (cm) -1 [gelatin] = 0.4 mg/ml a = (0.044 / 0.4 mg/ml · 1cm) = 0.11 (mg/ml) -1 (cm) -1 The A280 absorbance by proteins is based on the aromatic amino acid side chains absorbing UV light: primarily by tryptophan and tyrosine. Question 1 above shows tryptophan to have a 4-times greater molar absorptivity than tyrosine. On a mass basis, gelatin is 5-times more concentrated than lysozyme, yet shows ~4.6-times less absorbance; Table 2.1 shows there are no tryptophans and only 2 tyrosines out of the 213 amino acids per gelatin molecule; whereas lysozyme has 6 tryptophans and 3 tyrosines per 133 amino acids. Lysozyme has a much higher molar percentage of both W and Y than gelatin (what is the mole % of W and Y for BSA?). 3. Using the overlay spectra data obtained in Part B for the Bradford reagent and the Bradford reagent plus protein: provide a brief explanation for performing the Bradford protein assay at 595 nm. A protein assay can be developed anywhere there is a measurable change in absorbance between the
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Exp_2_KEY_W12 - Experiment 2 KEY MCB120L, W12 Overlay...

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