Lecture 6

Question 1

When storing proteins what can you use to keeps them stable

Answer 1

Chemical stabilizers

Question 2

What can proteins be stored as

Answer 2

Ammonium sulphate precipitate

A solutions with a protectant (20-50% glycerol)

Lyophilized (freeze dried)

Question 3

What is something to take into account when lyophilizing

Answer 3

When the protein gets thawed, It could potentially denature

Question 4

What are examples of chemical stabilizers and what they do

Answer 4

Polyols

Other proteins

Reducing agents for proteins without disulphide bonds

Question 5

Give examples of polyols and what they do

Answer 5

Glycerol, sucrose, polyethylene glycol

Increase the Tm

Question 6

How do proteins stabilize other proteins

Give an example

Answer 6

By interacting with each other through docking each other

BSA or oval decrease surface denaturation of proteins

Question 7

How do reducing agents stabilize proteins

Give examples

Answer 7

The help proteins that have unnatural disuphide bonds (normally shouldn’t have them) These bonds lead to aggregation

Some enzymes use S- (Thiolite anions) to regulated redox reactions

Ex. TCEP, 2ME

Question 8

Slide 4

Answer 8

Not in midterm

Question 9

What is Tm

Answer 9

The temperature at which [N]=[U]

N= native folded protein

U= unfolded protein

So if fraction of unfolded is 0.5 the temp at 0.5 is the melting point

Question 10

Tm of ribonuclease

Answer 10

30 degrees Celsius

Question 11

What enzymes are used in a restriction digest

Answer 11

HINDIII

ECORI

Question 12

What in the ECOR1 helps with restriction digest

Answer 12

KPO4 (the phosphate binds to the active site and holds the enzyme in a confirmation that keeps it happy)

EDTA (chelates metal ions)

Low concentration BSA

Question 13

What in the ECOR1 helps with restriction digest

Answer 13

KPO4 (the phosphate binds to the active site and holds the enzyme in a confirmation that keeps it happy)

EDTA

Question 14

What in HINDIII helps in restrictions digest

Answer 14

Higher BSA concentration

Question 15

After restriction digest what happens

Answer 15

The bases that are cut are put into gel electrophoresis

Larger dna fragment travel slow

Smaller travel faster

Question 16

Enzymes reduce the

Answer 16

Activation energy of a reaction (smaller peak)

Question 17

What is an example of are reaction that an enzyme catalyzes

Answer 17

Glycolysis, glucose to co2

This is respiration reaction

Question 18

What is michealis menten kinetics

Answer 18

Measure the reaction rate at different substrate concentrations but constant enzyme concentration

Constant pH, temp , ionic strength

Question 19

What is Vmax

Answer 19

In the menten plot it’s when the enzyme can’t convert substrate to product any faster

Question 20

What are the two types of enzyme assays

Answer 20

Direct : ex the substrate gets fe phosphorylated in the reaction and we detect the released phosphate

Indirect: coupled enzyme assay (the product in one reaction gets consumed in another reaction)

Question 21

Why would the product formed over time in a enzyme assay drop off at a certain time

Answer 21

The substrate concentration may have dropped

The feedback inhibition by Product that signals there’s enough so no more is made

Enzyme inactivation (they inactivate at too high of a temp)

Question 22

What is steady state catalysis

Answer 22

The concentration of the enzyme substrate complex is constant

So there is no change in the concentration of the enzyme substrate complex concentration

Question 23

What is the assumption of the substrate concentration during the initial rate period of enzyme reacting with substrate

Answer 23

The substrate concentration is constant

The concentration of substrate is much higher than enzyme concentration

This means the amount of S used in making the ES complex is basically zero

Meaning intially the change in S concentration is zero (since there’s already so much S)

Basically like no substrate was used up

Question 24

What reaction does G6PDH do and how do we measure it

Answer 24

Reduced NADP+ to NADPH

Can measure absorbace of NADPH at 340nm

Question 25

What is the extinction coefficient of NADPH and NADH

Answer 25

6.22mM-1cm-1

Question 26

What type of enzyme is G6PDH

Answer 26

A Pentose phosphate pathway enzyme

Question 27

In an assay what do we want the change in absorbace per min to be

Answer 27

0.01-0.1

Question 28

What happens to the change in A/min if the Enzymes concentration is too low What happens if the Enzymes concentration is too high

Answer 28

The rate is too small and this leads to large uncertainty If E too high our assumption of the S concentration being so much higher than E is wrong and the change in substrate concentration after enzyme uses it is not zero

Question 29

Slide 16 calc

Answer 29

Okay

Question 30

Slide 17 calc of enzyme velocity

Answer 30

Okay

Question 31

What is one Unit (U) of G6PDH defined as

Answer 31

Reducing one micromol of NADP per minute So activity in U = V0 in micromol/min

Question 32

What is specific activity

Answer 32

U/mg of enzyme

Question 33

How do you calculate specific activity of the enzyme

Answer 33

If diluted, account for dilution, then find mg of enzyme in solution by multiplying by the final volume it’s in Then do U/mg U is the initial velocity

Question 34

Slide 18 specific activity cal

Answer 34

Okay

Question 35

If you are doing a t test for two experiments with two separate means, how do you find the degrees of freedom

Answer 35

If 3 trials in each set Add # trail of first experminet and of second 6 Then minus 2 (because two means) 4 3+3-2=4

Question 36

When is the difference in two means significant

Answer 36

If the calculated t value is greater than the one in the table So if t stat is higher than t critical two tail