The pentapeptide Val-Arg-Gln-Trp-Cys has N-terminal and C-terminal pKas of 9.5 and 4.5, respectively. The side chain pKa's are Arg 12.5 and Cys 8.0.
a. Write this peptide sequence using single letter abbreviations.
b. Draw its predominant chemical structure at pH 7.4, including charges.
c. What fraction of the peptide will exist in the predominant form at pH 7.4?
d. Calculate the pI of the peptide.
e. If this pentapeptide is reacted with NEM (N-ethylmaleimide), what will be the pI of the product? f. To separate a mixture of the peptide and its NEM reaction product by ion exchange
chromatography at pH 7.4, would you choose a CM- or DEAE-matrix? Explain why.
g. Which peptide would elute first? Explain why.
h. If the peptide and its NEM reaction product were separated by ESI TOF MS, which would reach
the detector first? Explain why.
i. If a solution of the pentapeptide, pH 6, has an absorbance of 0.5 at 280 nm in a standard 1-cm width spectrophotometer cuvette, what is the molar concentration of the peptide? What is its concentration in mg/ml? (For an estimate of molar absorptivity ?, in units of M-1 cm-1, see G & G Fig. 4-10 and round to one significant figure; for Mr values of amino acids, see lecture slide 4- 18.)