1. Cytochrome C

Question 1

Why do tissues such as the heart contain an abundant supply of Cyt C?

Answer 1

[Look at PostLab answers lmao]

Question 2

What is ion exchange chromatography?

Answer 2

A chromatographic method of purification that relies on electrostatic attraction between oppositely charged molecules

Question 3

What are the two forms of ion-exchange resin commonly used in the purification of proteins (+ one example for each)?

Answer 3

ANION EXCHANGERS (e.g., DEAE-Cellulose) are positively charged at neutral pH and bind anions
CATION EXCHANGERS (e.g., CM-Cellulose) are negatively charged at neutral pH and bind cations

Question 4

Protonation and Deprotonation occur when WHAT kinds of buffers are added to a solution?

Answer 4

Protonation of anions occurs when a buffer with a LOWER pH THAN pK is added (add more H+, eqb shifts to left, A- -> HA)

Deprotonation of HA occurs when a buffer with a HIGHER pH THAN pK is added (less H+, eqb shifts to right, HA -> H+ + A-)

Question 5

What sort of ion exchange resin should be used to isolate a particular protein?

Answer 5

One which carries the OPPOSITE CHARGE to the protein

Question 6

Should DEAE-Cellulose or CM-Cellulose be used to purify cytochrome c?

Answer 6

Cytochrome c contains many basic AA residues, with a pKa of around 9; CM Cellulose/Sephadex side groups have a pKa of around 5;

So at pH 7, cyt c will be protonated (as pH below pKa), while CM Sephadex groups will be deprotonated (pH above pKa), so cyt c will bind to carboxyl side chains.

Therefore, a cation exchanger such as CM-Cellulose should be used.

Question 7

How is cytochrome c released (eluted) from the column, once it has bound to the CM Sephadex?

Answer 7

Buffer containing sodium chloride is added, thus increasing the ionic strength and eluting cyt c (even though the pH remains at 7.0)