The aromatic side chain of Trp residues in proteins and peptides can be chemically modified by two photochemical reactions using ultraviolet light (hv) at 20°C (Figure 1). hv CCI3CO₂H (10 mm), Buffer A (pH 8.0) HO HN HN- O B. Basic O A. Acidic NH Figure 1 Photochemical reactions of Trp side chain with CHCI, and CCl₂CO₂H NH ww Each substitution on Trp replaces one hydrogen atom attached to a carbon somewhere on the indole ring. O c. Hydrophobic OD. Polar neutral Photochemical modification percentages of various Trp residues in carbonic anhydrase at 20°C are shown in Table 1. Table 1 Photochemically Modified in Carbonic Anhydrase Reactant Buffer W4|W15|W96|W122|W190|W207|W243 CHCI3 A 37% 0% 0% 0% 51% 0% 44% CHCl3 B 0% 0% 0% 0% 0% 0% 55% CCI,CO₂H A 72% 66% 0% 100% 100% 0% 75% OA. Imidazole W15 and W96, which are in the interior of carbonic anhydrase, are 21% and 29% modified respectively by 10 mM CHCl3 in Buffer A at 75°C, a temperature which fully denatures carbonic anhydrase but leaves its primary structure intact. Access to W15 in fully folded carbonic anhydrase is blocked by two His residues and one Lys residue. Like the unmodified enzyme, photochemically modified carbonic anhydrase at 20°C has full enzyme activity, which is the conversion of CO₂ to H₂CO3. Native carbonic anhydrase also has a net charge of -2.9 at pH 8.0. OB. Indole O c. Pyrimidine OD. Pyrrole hv Adapted from C. L. Ladner, R. J. Turner, and R. A. Edwards, Protein Science. C2007 The Protein Society. Which classification of amino acids applies to the Trp residues after photochemical modification by CCI,CO₂H? CHCl3 (10 mM), Buffer A (50 mM NH4HCO3, pH 8.0) or Buffer B (50 mM NH4CH3CO₂, pH 6.5) Which type of heterocycle is found on two amino acid residues blocking access to W15? O A. Ammonium formate Based on the passage, what most likely causes W96 to be accessible to CHCI, at 75°C? O A. Peptide bonds are broken, releasing W96. OB. Reduction of disulfide bonds occurs. OC. The protein unfolds and exposes W96 to the buffer. OD. CHCl3 extracts W96 from the protein interior. OB. Ammonium carbonate What is the name of the ionic compound used to make Buffer B? OC. Ammonium bicarbonate O D. Ammonium acetate HN- Which chromatographic technique would most likely separate a mixture of native carbonic anhydrase from carbonic anhydrase photochemically modified by CCI,CO₂H? OA. Anion-exchange chromatography OB. Cation-exchange chromatography O c. Gas-liquid chromatography OD. Size-exclusion chromatography Which Trp residue of carbonic anhydrase can be selectively modified with CHCl, at 20°C? O A. W4 O B. W96 O C. W190 O D. W243