Lecture 6 Flashcards
When storing proteins what can you use to keeps them stable
Chemical stabilizers
What can proteins be stored as
Ammonium sulphate precipitate
A solutions with a protectant (20-50% glycerol)
Lyophilized (freeze dried)
What is something to take into account when lyophilizing
When the protein gets thawed, It could potentially denature
What are examples of chemical stabilizers and what they do
Polyols
Other proteins
Reducing agents for proteins without disulphide bonds
Give examples of polyols and what they do
Glycerol, sucrose, polyethylene glycol
Increase the Tm
How do proteins stabilize other proteins
Give an example
By interacting with each other through docking each other
BSA or oval decrease surface denaturation of proteins
How do reducing agents stabilize proteins
Give examples
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
Slide 4
Not in midterm
What is Tm
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
Tm of ribonuclease
30 degrees Celsius
What enzymes are used in a restriction digest
HINDIII
ECORI
What in the ECOR1 helps with restriction digest
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
What in the ECOR1 helps with restriction digest
KPO4 (the phosphate binds to the active site and holds the enzyme in a confirmation that keeps it happy)
EDTA
What in HINDIII helps in restrictions digest
Higher BSA concentration
After restriction digest what happens
The bases that are cut are put into gel electrophoresis
Larger dna fragment travel slow
Smaller travel faster
Enzymes reduce the
Activation energy of a reaction (smaller peak)
What is an example of are reaction that an enzyme catalyzes
Glycolysis, glucose to co2
This is respiration reaction
What is michealis menten kinetics
Measure the reaction rate at different substrate concentrations but constant enzyme concentration
Constant pH, temp , ionic strength
What is Vmax
In the menten plot it’s when the enzyme can’t convert substrate to product any faster
What are the two types of enzyme assays
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)
Why would the product formed over time in a enzyme assay drop off at a certain time
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)
What is steady state catalysis
The concentration of the enzyme substrate complex is constant
So there is no change in the concentration of the enzyme substrate complex concentration
What is the assumption of the substrate concentration during the initial rate period of enzyme reacting with substrate
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
What reaction does G6PDH do and how do we measure it
Reduced NADP+ to NADPH
Can measure absorbace of NADPH at 340nm
What is the extinction coefficient of NADPH and NADH
6.22mM-1cm-1
What type of enzyme is G6PDH
A Pentose phosphate pathway enzyme
In an assay what do we want the change in absorbace per min to be
0.01-0.1
What happens to the change in A/min if the Enzymes concentration is too low
What happens if the Enzymes concentration is too high
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
Slide 16 calc
Okay
Slide 17 calc of enzyme velocity
Okay
What is one Unit (U) of G6PDH defined as
Reducing one micromol of NADP per minute
So activity in U = V0 in micromol/min
What is specific activity
U/mg of enzyme
How do you calculate specific activity of the enzyme
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
Slide 18 specific activity cal
Okay
If you are doing a t test for two experiments with two separate means, how do you find the degrees of freedom
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
When is the difference in two means significant
If the calculated t value is greater than the one in the table
So if t stat is higher than t critical two tail
