Lecture 3

Question 1

What is the equation for absorbance?

Answer 1

A = log(Io/I) = e* l * c

Io = intensity of incident light
I = intensity of transmitted light
e = absorptivity
l = path length
c = concentration

Question 2

Describe how absorbance spectroscopy is used to measure protein concentration.

Answer 2

A single wave length of light (produced either by LED or light passing through monochromator) is shone through sample. The initial intensity of the light is known and the intensity of light that is transmitted through the sample is measured at a detector. This relationship is used to calculate the absorbance of the sample. Amino acids (and thus proteins as a whole) have characteristic wavelengths at which they absorb light. Thus, by knowing the absorbance the concentration can be found using beer’s law (A=elc)

Question 3

Describe salting out.

Answer 3

Salting out is used to crudely precipitate proteins. Salt can be added in an increasing concentration. With increasing [salt], the salt will interact with water molecules in solution and with any charged or polar groups on the surface of the protein. This will cause less water molecules to be able to interact with the protein and will also neutralize the surface charges on the protein. As the proteins become neutralized they will clump together with one another and fall out of solution. Because different proteins have different amounts of charge on their surface, the proteins in solution will precipitate at different [salt].

Question 4

What are the general steps to chromatography? (Specifically, ion exchange)

Answer 4

1) determine pH of buffer (based on protein of interest and bead type)
2) rum buffer through column to make beads charged at desired buffer pH
3) load sample
4) wash with buffer
5) begin salt or H+ gradient to elute proteins
6) collect fractions

Question 5

What is specific activity?

Answer 5

enzyme units per mg of total protein

Increases with greater purification, becomes maximal and constant when enzyme is pure

Question 6

Once specific activity is calculated, how are specific activities compared?

Answer 6

The purification factor is calculated. If I have a 4 step purification method, then the purification factor of step 1 is always 1. Then for step 2, I divide the SA of step 2 by the SA of step 1. I continue in this fashion for all steps. Let’s say step 3 / step 2 = 10 and that is the largest purification factor. That would mean step 2 was the most effective step at purifying my sample. Let’s say step 2/1 is 0.5. That would mean step 1 is least effective in purifying my sample and actually results in a loss of purification.

Question 7

What 4 things does SDS do?

Answer 7

1) denature proteins
2) confer net negative charge to protein
3) disrupt non covalent bonds
4) disrupts conformation

Question 8

I’m SDS PAGE, how do proteins separate?

Answer 8

By size, large proteins travel slower through gel because they cannot pass as quickly through porous acrylimide, smaller proteins are down toward the bottom.

Question 9

Describe western blot.

Answer 9

SDS PAGE is done first to separate by size. Protein bands from gel are transferred to a membrane. Membrane is incubated with primary antibody that only recognizes protein of interest. Membrane is incubated with secondary antibody that recognizes constant region of primary antibody. Secondary antibody is radio labeled or contains substrate for colored precipitate reaction.

Question 10

How is composition of protein determined via chromatography in general?

Answer 10

1) polypeptide is hydrolyzed (acid, base, enzymatic)
2) free AAs are tagged with fluorescent label
3) AAs identified by characteristic retention times on column chromatography compares to standards
4) amount of AA is quantified by fluorescence

Question 11

What steps must happen in order to sequence a protein?

Answer 11

1) denature protein (disrupt non covalent bonds and break di sulfide bonds)
2) cleave proteins into smaller peptides using enzymes that cleave protein at different locations along peptide chain
3) sequence protein from each enzymatic digestion separately and compare overlapping parts of sequence to arrive at sequence of AAs

Question 12

What is Edman degradation?

Answer 12

A means of sequencing peptides. Edman’s reagent binds only to the n terminus, then it catalyzes the breaking of the bond between the 1st and 2nd AA. Once the 1st AA is free, that can be sequenced by chromatography. The 2nd round the reagent will bind to AA 2 and break the bond between AA 2 and 3. The process repeats in that manner.

Question 13

Why do we need hemoglobin and myoglobin?

Answer 13

O2 is essential for cellular respiration and survival. It is non polar and dissolves poorly in blood and cannot diffuse over large distances. Therefore, hemoglobin is needed to transport O2 and myoglobin is needed to store O2.

Question 14

Heme:

1) how many bonds to Fe?
2) what must be the oxidation state of Fe?

Answer 14

1) 6
4 to porphyrin ring
1 to histidine of protein
1 to O2

2) Fe2+ (Fe3+ cannot bind O2)

Question 15

What is p50 as it relates to myoglobin and hemoglobin?

Answer 15

Measure of the affinity of protein for oxygen, lower p50 means takes less O2 to bind to 50% of protein, thus low p50 means high affinity

Question 16

Describe the T and R states of hemoglobin.

Answer 16

The T state is when hemoglobin is deoxygenated and reluctant to bind more oxygen. This reluctance stems from the fact that the heme group on the porphyrin ring is buried within the subunit of the protein and in the tense conformation the heme in the rings are positioned such that it is not favorable for O2 to bind. Once 1 O2 binds, that causes a conformational change in another subunit of the protein such that the porphyrin ring shifts so the heme Group is better positioned in that subunit to bind O2. This conformational change in favor of binding O2 continues and progresses the hemoglobin to the R state (oxygenated Hb).

Question 17

How does H+ affect hemoglobin bonding to O2?

Answer 17

Increased [H+] = decreases affinity of Hb for O2.

H+ binds to residues causing formation of ionic bonds between residues. This increases stability of Hb makes Hb more rigid, favoring the conformation of the T state, and promoting the dissociation of O2 from Hb. This occurs naturally in tissue capillaries because O2 needs to be released to tissues and H+ needs to be transported out of tissues.

Question 18

Effect of CO2 on Hb affinity for O2?

Answer 18

CO2 binds to n-termini of globin chains creating carbamate ion which can form ionic bonds with other residues. These bonds stabilize the Hb and make the structure more rigid, favoring the T state and thus dissociation of O2. Also occurs naturally in tissue capillaries.

Question 19

What effect does BPG have on Hb?

Answer 19

Increased BPG decreases Hb affinity for O2. BPG is naturally produced in RBCs as a control measure for Hb affinity to O2. It is able to alter Hbs affinity for O2 in response to changes in available pO2 in the lungs. It binds to the subunits is Hb in the center of the tetramer. This central core is only accessible in the t state so BPG binding favors the T state conformation and thus decreased affinity of Hb for O2. This makes little difference in lungs where pO2 is high, but a big difference in tissues where pO2 is low. Results in increased release of O2 in tissues.