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3. (a) In Bakers yeast there are two variants of cytochrome c, a heme containing protein of 108 amino acid residues that catalyzes electron transfer reactions in mitochondria. The two forms of cyto- chrome c known as iso-1 and iso-2 cytochrome c differ in 17 of the 108 amino acids. The content of proton dissociable side chains for the two cytochromes c is given in the table below: Side chain pka iso-1 cytochrome c Ziso-1 iso-2 cytochrome c 12.5 3 4.0 4 8.0 1 4.0 4 6.0 4 10.0 16 9.0 7.0 4.0 Arginine Aspartate Cysteine Glutamate Histidine Lysine Tyrosine -NH3Ⓡ -COOH 5 1 1 What is the overall electrostatic charge on each cytochrome at pH 8? (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 of NaCl applied to the column, which ion Na or Cl displaces the proteins from the binding sites on the chromatographic resin? 0 0 carboxymethyl cellulose OH 0 350737511 CH 3 ΘΝΗ HO Ziso-2 HỌC NH H3C₁ 2-(diethylamino)ethyl cellulose (DEAE cellulose) (c) Which protein iso-1-cytochrome c or iso-2-cytochrome c will be the first to be eluted from the column and why? (d) 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 will be the overall electrostatic charge on the peroxidase enzyme for this reaction to occur, positive or negative?

3. (a) In Bakers yeast there are two variants of cytochrome c, a heme containing protein of 108 amino acid residues that catalyzes electron transfer reactions in mitochondria. The two forms of cyto- chrome c known as iso-1 and iso-2 cytochrome c differ in 17 of the 108 amino acids. The content of proton dissociable side chains for the two cytochromes c is given in the table below: Side chain pka iso-1 cytochrome c Ziso-1 iso-2 cytochrome c 12.5 3 4.0 4 8.0 1 4.0 4 6.0 4 10.0 16 9.0 7.0 4.0 Arginine Aspartate Cysteine Glutamate Histidine Lysine Tyrosine -NH3Ⓡ -COOH 5 1 1 What is the overall electrostatic charge on each cytochrome at pH 8? (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 of NaCl applied to the column, which ion Na or Cl displaces the proteins from the binding sites on the chromatographic resin? 0 0 carboxymethyl cellulose OH 0 350737511 CH 3 ΘΝΗ HO Ziso-2 HỌC NH H3C₁ 2-(diethylamino)ethyl cellulose (DEAE cellulose) (c) Which protein iso-1-cytochrome c or iso-2-cytochrome c will be the first to be eluted from the column and why? (d) 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 will be the overall electrostatic charge on the peroxidase enzyme for this reaction to occur, positive or negative?

Biochemistry
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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### Educational Content: Cytochrome c Variants in Bakers Yeast #### 3. (a) Cytochrome c Variants In Bakers yeast, two variants of cytochrome c exist, known as iso-1 and iso-2. Each variant is a heme-containing protein with 108 amino acids that facilitates electron transfer in mitochondria. These variants differ in 17 amino acids. The table below shows proton dissociable side chains and their content in both cytochrome c variants: | Side Chain | pKa | iso-1 cytochrome c | \( Z_{\text{iso-1}} \) | iso-2 cytochrome c | \( Z_{\text{iso-2}} \) | |------------|------|--------------------|-----------------|--------------------|-----------------| | 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 | | –NH₃⁺ | 7.0 | 1 | 1 | 1 | 1 | | –COOH | 4.0 | 1 | 1 | 1 | 1 | *Question: What is the overall electrostatic charge on each cytochrome at pH 8?* #### (b) Ion Exchange Chromatography The diagram illustrates chemical groups in carboxymethyl-cellulose and DEAE-cellulose, used for ion exchange chromatography in protein purification: - **Carboxymethyl-cellulose**: Negatively charged at pH 8. - **DEAE
Transcribed Image Text:### Educational Content: Cytochrome c Variants in Bakers Yeast #### 3. (a) Cytochrome c Variants In Bakers yeast, two variants of cytochrome c exist, known as iso-1 and iso-2. Each variant is a heme-containing protein with 108 amino acids that facilitates electron transfer in mitochondria. These variants differ in 17 amino acids. The table below shows proton dissociable side chains and their content in both cytochrome c variants: | Side Chain | pKa | iso-1 cytochrome c | \( Z_{\text{iso-1}} \) | iso-2 cytochrome c | \( Z_{\text{iso-2}} \) | |------------|------|--------------------|-----------------|--------------------|-----------------| | 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 | | –NH₃⁺ | 7.0 | 1 | 1 | 1 | 1 | | –COOH | 4.0 | 1 | 1 | 1 | 1 | *Question: What is the overall electrostatic charge on each cytochrome at pH 8?* #### (b) Ion Exchange Chromatography The diagram illustrates chemical groups in carboxymethyl-cellulose and DEAE-cellulose, used for ion exchange chromatography in protein purification: - **Carboxymethyl-cellulose**: Negatively charged at pH 8. - **DEAE
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