A peptide mixture contains the following three peptides: Peptide 1: Val-Glu-Glu-

Question

A peptide mixture contains the following three peptides: Peptide 1: Val-Glu-Glu-Glu-Ser-His-Lys-Gly-Trp-Thr Peptide 2: Val-Gin-Gin-Gln-Ser-His-Lys-Gly-Trp-Thr Peptide 3: Glu-Glu-Glu-His-Lys A solution of the peptide mixture at a pH value of 6.0 is prepared. 1) Estimate the charges of the peptides in the solution. (pK values: N-terminus: 9, C terminus: 2, lysine: 10, arginine 12, aspartic acid: 3, glutamic acid: 4, histidine: 6, cysteine: 8, tyrosine: 10) 2) If the peptides were separated by using an anion exchange column at a pH of 6.0 and a linear salt concentration gradient, what would be the order of elution? Justify your answer

Explanation / Answer

the net charges on peptide 1,2 and 3 are -2, 1.5 and -1.5 respectively.

in anion exchange chromatography negatively charged peptide will bind strongly. so the order of elution is-

peptide 2 then peptide 3 and last peptide 1.

in cation exchange chromatography positively charged peptide will bind strongly. so the order of elution is-

peptide 1 then peptide 3 and last peptide 2.

gel filtration chromatography separate peptide on the basis of size so larger size will elute first and smaller size will elute last. since peptide 1 and 2 have approx similar size so they coelute and peptide 3 will elute at the end.

first we will perform gel filtration to separate 3 from peptide 1 and 2. then we load the coeluted peptide 1 and 2 on any ion exchange column.

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