1 Identify the peaks in the chromatogram in Figure 183 using

30 pH 6.5 20 10 pH 5.0 20 30 50 40 60 Fraction numbe Activity pH Figure 18.3: Cation exchange chromatography of PFK-1 (based on Foe and Kemp, 1985). 3. Next, the investigators took Fractions 16-26 from the DEAE-Sephadex column, pooled them, and adjusted the pH to 5.0. This preparation was then loaded onto a CM-52 (cation exchange) chromatography column and eluted with a pH gradient. Fractions 30-35 were collected and pooled, as were Fractions 41-50. Identify the peaks in the chromatogram in Figure 18.3, using information in the SDS-PAGE gel. Based on their clution from the cation exchange column, how might the amino acid compositions of these two proteins differ?

Solution

1) The first peak in the chromatogram from the left represents the fraction of the protein which contains anions ie aspartic acid and glutamic acid residues mainly. And the 2nd peak represents the protein fragment that contains positively charged residues like lysine and arginine.

This can be deduced because it is a cation exchange coloumn and negatively charged amino acids will be eluted first and the positively charged cations in amino acids lysine and arginine will be eluted at the end.

2)From the SDS PAGE data we can deduce the nature of the protein PFK . Lane 3 &4 contains pooled fractions of regions 16-26 & 55-60 respectively . DEAE sephadex coloumn is an anion exchanger so we can conclude the residuess 16-26& 55-60 of the protein mainly consists of negatively charged amino acid like aspartic acid and glutamic acid . Whereas lanes 5&6 contains pooled fractions of residues 30-35 & 41-50 respectively. CM52 is a cation exchanger so we can conclude that residues  30-35 & 41-50 mainly consists of positively charged amino acids lysine and arginine.