Quiz 3: Using Gel Filtration to Study Ligand-Protein Interactions

Chemistry

In this problem, we are asked to explain the elution patterns of the following amino acids on a cation-exchange chromatography column, set to a pH of 4. a) Gly, Asp, His b) Arg, Glu, Ala c) Phe, His, Glu The order of elution at pH 4 is Cys-A, Asn, Gln, Ser, Thr, Asp, Gly, His, Glu, Ala, Arg, Cys, Tyr, Pro, Val, Met, Ile, Phe, Leu, nor-Leu, Trp, Lys. This order is based on the side-chains of the amino-acids, which will either gain or lose protons based on their acidic or basic natures and the pH of the column. a)The order would be Asp, Gly, His. b)The order would be Glu, Ala, Arg. c)The order would be His, Glu, Phe.

In this problem, we are asked to explain the relative R f values for the following amino acids in a paper chromatograph - Ser, Lys, Leu, Val, and Ala. The film has been developed with n -butanol, water, and acetic acid. The nature of the paper and the solvent will affect the movement of these different amino acids based on their side-chains. The paper is more basic, while the solvent used here is acidic (the acetic acid) and slightly less polar than pure water due to the presence of the n -butanol. Considering the side-chain interactions of the various amino acids in this given set-up, the pattern of elution will be Ser, Lys, Ala, Val, Leu.

In this problem, we are asked to explain the order of elution from a DEAE-cellulose ion exchanger with an increasing salt gradient for the following compounds with their pH 1. a) Egg albumin, 4.6 b) Cytochrome c , 10.6 c) Pepsin, 1.0 d) Myoglobin, 6.8 e) Hemoglobin, 6.8 f) Serum albumin, 4.9 DEAE set-ups use salts to modulate the pH of the mobile phase. Materials in the solution either remain on the column or elute as the salt gradient increases. Because of this principle, the compounds elute in order from decreasing pH as the salt gradient increases. Thus the order will be cytochrome c , followed by myoglobin and hemoglobin together, then serum albumin, then egg albumin, then pepsin.

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