Protein Purification & RuBisCo

Question 1

What does RuBisCo stand for?

Answer 1

Ribulose-1,5-bisphosphate carboxylase/oxygenase

Question 2

_________________ is the most abundant protein in chloroplasts and makes up approximately 50% of protein in plant leaves

Answer 2

RuBisCo

Question 3

What does RuBisCo do?

Answer 3

Fixes CO2 to a two 3C molecules of 3-phosphoglycerate via a short-lived 6C intermediate ribulose-1,5-bisphosphate

Question 4

RuBisCo fixes CO2 to a two 3C molecules of ____________________ via a short-lived 6C intermediate __________________

Answer 4

3-phosphoglycerate

Ribulose-1,5-bisphosphate

Question 5

RuBisCo contains __________________ structure as it’s composed of eight large and eight small subunits

Answer 5

Quaternary

Question 6

What’s the total enzyme size of RuBisCo?

Answer 6

560 kDa

Question 7

The ___________________ has 1/12 the mass of an unbound atom of C12

Answer 7

Dalton

Question 8

How much mass does a Dalton contain?

Answer 8

1.66 x 10^-27 kg

Question 9

Two ways to precipitate proteins were discussed in class. What are they?

Answer 9

Fractional precipitation

Salting In/Salting Out

Question 10

In ____________________ _______________ of proteins, either the contaminants or proteins of interest precipitate and undergo chromatographic analysis

Answer 10

Fractional precipitation

Question 11

What technique separates ions from solution based on their different solubilities?

Answer 11

Fractional precipitation

Question 12

At _______ salt concentrations, protein solubility increases because salt prevents charge-charge interactions between proteins

Answer 12

Low

Question 13

_________ salt concentrations prevent protein aggregation and thus precipitation

Answer 13

Low

Question 14

At ______ salt concentrations, protein solubility decreases because salt competes with solutes for solvent

Answer 14

High

Question 15

________ salt concentration removes the solvent from proteins, and they “crash” out of solution

Answer 15

High

Question 16

What salt did we use in experimentation?

Answer 16

Ammonium sulfate

Question 17

Why did we use ammonium sulfate?

Answer 17

At saturation, it has a high enough molarity to precipitate most if not all proteins

Question 18

Besides its high molarity at saturation, why else did we use ammonium sulfate?

Answer 18

It does not have a high heat of solution, so heat can be easily distributed; it precipitates most proteins without affecting protein ability to sediment via centrifugations; it prevents bacterial growth at high concentrations; and it protects proteins from denaturation

Question 19

The concentration of ammonium sulfate required to precipitate a protein varies from protein to protein and is thus _________________ determined

Answer 19

Empirically

Question 20

The more ____________________ exposed side chains of a protein, the more salt will be required to salt the protein out of solution

Answer 20

Hydrophilic

Question 21

Generally ________________ (larger or smaller) proteins need less salt to precipitate out of solution

Answer 21

Larger

Question 22

Which will require more salt to precipitate: a protein with many exposed hydrophilic or hydrophobic residues?

Answer 22

Hydrophilic

Question 23

Which will require more salt to precipitate: a larger or smaller protein?

Answer 23

Larger

Question 24

What are two methods of salt removal discussed in class?

Answer 24

(1) Desalting columns

(2) Dialysis

Question 25

Desalting uses the principle of ______________________ __________________

Answer 25

Size exclusion

Question 26

In a desalting column, the pores are made slightly smaller than the target protein so that it will elute with the ___________ phase and the salt will be trapped in the ___________ phase

Answer 26

Mobile | Stationary

Question 27

Explain the principle of size exclusion in desalting.

Answer 27

In desalting, there is a large size difference between the target molecules in the sample and unwanted salt or small molecules. A size exclusion limit is chosen that is lower than the size of the target molecules (that is, target molecules are too big to enter the pores of the beads). Therefore, they are excluded from the pores, remain in the mobile phase, and are eluted after the void volume. Salts and other small molecules are retarded by the desalting column and are thereby separated from the target molecules.

Question 28

_________________ is one of the most frequently used separation techniques to facilitate the removal of small, undesired materials from macromolecules in solution by a selective semi-permeable membrane

Answer 28

Dialysis

Question 29

How does dialysis work?

Answer 29

The size of pores of these semi-permeable membranes determine their molecular-weight cutoff. The principle of dialysis is that sample molecules will be retained on the sample side since they are larger than the membrane pores, while small molecules diffuse freely to reach an equilibrium concentration. As a result, the concentration of small unwanted molecules decreases to negligible levels, while the concentration of the sample increases to desired levels.

Question 30

In dialysis, which leaves the dialysis bag: the protein of interest or salt?

Answer 30

Salt because it is small enough to leave

Question 31

In dialysis, when does the biggest drop in solute concentration occur?

Answer 31

During the first hour or so with a gradual decrease until equilibrium is reached