Question

A protein known to be stably folded at high pH titrated from pH 8 to pH 11 with spectra recorded at intervals of 0.1 units/ The molar extinction coefficient at 295 nm increases sharply at pH 9.7.

a) What does this suggest about the amino acid composition of the protein? Why?

b) What does this suggest about the location of specific titratable groups in the protein? Why?

Answer 1

a) Absorption of UV-visible light of the range 200-300 nm by proteins is mainly by the aromatic side chain residues of the amino acids.These are the residues of tryptophan (trp),tyrosine(tyr) or phenylalanine(Phe) with aromatic side chain.

But the typical absorption at 295 showing elevated absorption at pH=9.7 is due to ionization of phenolic group of tyrosine (tyr) ,it has a pka=9.1,so it is deprotonated at high pH =9.7 and thus the phenolic group resonate at the said wavelength showing maxima.

b) This shows that tyr residues and mostly the phenolic group are not free to ionize/absorb in the native protein due to its compact folding in tertiary protein structures.When the protein denatures ,the tyr residue is unfolded to give maximum absorbtion.