(b) To the right is a diagram of the chemical groups in carboxymethyl-cellulose and DEAE-cellulose that are used for ion exchange chromatography to purify proteins. At pH 8 which chromatographic resin has to be used to separate iso-1-cytochrome c from iso-2- cytochrome c from a lysate of yeast cells? If the proteins are separated by application of a concentration gradient carboxymethyl of NaCl applied to the column, which ion Na® or Cle dis- places the proteins from the binding sites on the chroma- tographic resin? cellulose OH H3C CH3 ΘΝΗ HO HỌC, NH H3C 2-(diethylamino)ethyl cellulose (DEAE cellulose) Which protein iso-1-cytochrome c or iso-2-cytochrome c will be the first to be eluted from the column and why? Both cytochromes in their ferrous form are substrates of yeast cytochrome c peroxidase (CCP) and react optimally with this enzyme at pH 8 forming a Michaelis complex of 1:1 stoichiometry (CcP:cyt c). If the isoionic point of the peroxidase is 5.25, what must be the overall electrostatic charge on the peroxidase enzyme for this reaction to occur, positive or negative?
(b) To the right is a diagram of the chemical groups in carboxymethyl-cellulose and DEAE-cellulose that are used for ion exchange chromatography to purify proteins. At pH 8 which chromatographic resin has to be used to separate iso-1-cytochrome c from iso-2- cytochrome c from a lysate of yeast cells? If the proteins are separated by application of a concentration gradient carboxymethyl of NaCl applied to the column, which ion Na® or Cle dis- places the proteins from the binding sites on the chroma- tographic resin? cellulose OH H3C CH3 ΘΝΗ HO HỌC, NH H3C 2-(diethylamino)ethyl cellulose (DEAE cellulose) Which protein iso-1-cytochrome c or iso-2-cytochrome c will be the first to be eluted from the column and why? Both cytochromes in their ferrous form are substrates of yeast cytochrome c peroxidase (CCP) and react optimally with this enzyme at pH 8 forming a Michaelis complex of 1:1 stoichiometry (CcP:cyt c). If the isoionic point of the peroxidase is 5.25, what must be the overall electrostatic charge on the peroxidase enzyme for this reaction to occur, positive or negative?
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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Transcribed Image Text:## Cytochrome c Variants in Bakers Yeast
In Bakers yeast, two variants of cytochrome c, a heme-containing protein consisting of 108 amino acid residues that facilitate electron transfer reactions, have been identified. These variants, known as iso-1 and iso-2 cytochrome c, differ at 17 of the 108 amino acids. The table below details the content of proton dissociable side chains for the two cytochromes:
### Table of Proton Dissociable Side Chains
| Side Chain | pKa | iso-1 cytochrome c | Z<sub>iso-1</sub> | iso-2 cytochrome c | Z<sub>iso-2</sub> |
|------------|------|--------------------|------------------|--------------------|------------------|
| Arginine | 12.5 | 3 | 3 | 3 | 3 |
| Aspartate | 4.0 | 4 | 4 | 5 | 5 |
| Cysteine | 8.0 | 1 | 1 | 0 | 0 |
| Glutamate | 4.0 | 4 | 4 | 7 | 7 |
| Histidine | 6.0 | 4 | 4 | 3 | 3 |
| Lysine | 10.0 | 16 | 16 | 17 | 17 |
| Tyrosine | 9.0 | 5 | 5 | 5 | 5 |
| –NH<sub>3</sub><sup>+</sup> | 7.0 | 1 | 1 | 1 | 1 |
| –COOH | 4.0 | 1 | 1 | 1 | 1 |
### Chromatographic Separation
The diagram to the right illustrates the chemical groups in carboxymethyl-cellulose and DEAE-cellulose, which are used for ion exchange chromatography to purify proteins.
At a pH of 8, selecting the appropriate chromatographic resin is crucial for separating iso-1-cytochrome c from iso-2-cytochrome c sourced
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