Chromatographic Methods Three polypeptides, the sequences of which are represent

Question

Chromatographic Methods Three polypeptides, the sequences of which are represented below using the one-letter code for their amino acids, are present in a mixture:

ATKNRASCLVPKHGALMFWRHKQLVSDPILQKRQHILVCRNAAG

GPYFGDEPLDVHDEPEEG

PHLLSAWKGMEGVGKSQSFAALIVILA

Of the three, which one would migrate most slowly during chromatography through:

(a) an ion-exchange resin; beads coated with positively charged groups?

(b) an ion-exchange resin; beads coated with negatively charged groups?

(c) a size-exclusion (gel-filtration) column designed to separate small peptides such as these?

(d) Which peptide contains the ATP-binding motif shown in the following sequence logo?

Explanation / Answer

pH > pKa, "everything" will be in the deprotonated form. For an individual amino acid, "everything" is composed of the following: Amino Group, Carboxyl Group and side chain

Carboxyl Group has a pKa of about 5, at 7 the Carboxyl group will be DEPROTONATED (-1 charge)
Amino Group has a pKa of about 9-10, at pH 7 the Amino group will be PROTONATED (+1 charge)

Ala - Thr - Lys - Asn - Arg - Ala - Ser - Cys - Leu - Val - Pro - Lys - His - Gly - Ala - Leu - Met - Phe - Trp - Arg - His - Lys - Gln - Leu - Val - Ser - Asp - Pro - Ile - Leu - Gln - Lys - Arg - Gln - His - Ile - Leu - Val - Cys - Arg - Asn - Ala - Ala - Gly

At pH 7 net charge of above sequence is 7.2

2. GPYFGDEPLDVHDEPEEG

Gly - Pro - Tyr - Phe - Gly - Asp - Glu - Pro - Leu - Asp - Val - His - Asp - Glu - Pro - Glu - Glu - Gly

At pH 7 net charge of above sequence is -6.9

3. PHLLSAWKGMEGVGKSQSFAALIVILA

Pro - His - Leu - Leu - Ser - Ala - Trp - Lys - Gly - Met - Glu - Gly - Val - Gly - Lys - Ser - Gln - Ser - Phe - Ala - Ala - Leu - Ile - Val - Ile - Leu - Ala

at pH 7 net charge will be 1.1

a)

An ion exchange resin with positively charge group will stick negetively charged ion so in our case 1st and 3 rd sequence are positively charge they will not stick on resin but second will stick. among 1st and 2nd later has less pH so 1st peptide will come first then second last only 3rd will come

b)

An ion exchange resin with negatively charge group will stick positively charged ion so sequence last one will be 1st which has more positive charge. in resin 2nd peptide sequence will come first . then 3rd will come last only first peptide sequence will come

c)

among this second one is small peptide so we can separate second one with first using size exclusion gel filtration

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