Practicals Flashcards
What is the basis of the salting out principle?
How does ammonium sulphate allow for the salting out of ovalbumin?
What does sulphuric acid allow for?
What happens at this pH?
How does this occur?
What property of the reaction allows for absorbance to be measured?
Therefore what does this say about the efficiency of the reaction?
Which denaturant produces the higher number of moles of thiols for the concentration of denaturant? Guanidine-HCl or urea?
Reducing the solubility of a protein in a very high ionic strength solution such that it precipitates into an organic solute
Ammonium sulphate is a salt & in high concentration which attracts solvent molecules via ionic charges, such that protein-protein interactions are stronger than protein-solvent interactions resulting in precipitation
Reduces the pH of the solution to the pI of ovalbumin such that the net charge of the protein is 0
Ovalbumin precipitates
Attractive forces of both +ve and -ve mean that the protein aggregates with other protein molecules via attractive ionic forces
5,5’-dithio-bis(2-nitrobenzoate) DTNB reaction with ovalbumin gives rise to protein thiol and nitromercaptobenzoate ions which are yellow in colour
More the protein has denatured, the more thiols will be present and a stronger yellow colour & higher absorbance
Gu-HCl
What is the Km equation?
What is the Vmax equation?
What is the rate limiting step?
What is the equation for the turnover number?
What is the rate of formation of [ES] when steady state is assumed and why?
What is the Vo equation at steady state?
What does steady state mean?
(k-1+k2)/k1
Kcat x Eo
K2 (Kcat)
Kcat/Km
= 0 because at > 1
Vmax / 1 + Km/S
E + S <=> ES is at equilibrium so K1 = K-1
What are the 4 assumptions of Michaelis Menten?
What are the assumptions of So in steady state?
What is the burst phase?
How do you know when the burst phase is over?
At steady state what is assumed and why?
What can be extracted from the MM graph when [S]o is low?
What can be extracted from the MM graph when [S]o is high?
What is the x & y axis of MM graph?
- Don’t consider [S] depletion with Vo
- Don’t consider back reaction of product so no K-2
- Ignore inhibition of enzyme by the product
- Ignore time - low enzyme stability & exogenous factors
[So]»[E] as S is not a constant so S(t) = So
When [ES] builds up quickly
MM graph plateau & reach steady state
ES is not a function of t so d[ES]/dt = 0
Gradient = Vmax/Km
Vmax from the plateau
x = [So] mM and y = Vo mM/s
What is the gradient of the graph in MM & lineweaver burk?
From what equation can the lineweaver burk equation be derived?
What is the lineweaver burk equation?
What is the x & y axis of LB graph?
What are the x and y intercept?
MM = Vmax/Km (at low [S]) and LB = Km/Vmax
Vo = Vmax / 1 + Km/S
1/Vo = Km/Vmax x 1/S + 1/Vmax
x = 1/So and y = 1/Vo
X intercept = -1/Km and y intercept = 1/Vmax
What kind of rate constant is Kcat/Km?
What does it show in the limit of [So]«Km?
How can some enzymes have a faster diffusion limit than Kcat/Km?
What are 5 assumptions of MM?
Second order
How quickly enzyme & substrate come together to form ES
Electrostatic Steering
- [P]o = 0
- [S]»[E]
- [S] = [S]o
- at > 1 after the burst phase where a = k1s + k-1 + k2
- d[ES]/dt = 0 = at steady state after the burst phase
What is the equation for Vmaxapp from the general regulator?
How can you get Kcatapp from this?
What is Kmapp from the general regulator?
What is the feature of a competitive inhibitor?
What is the Kmapp of a competitive inhibitor going to be and why?
What is the Kmapp equation for a competitive inhibitor?
What is the Kcatapp of a competitive inhibitor going to be?
What does this show about Vmaxapp?
(Eo x Kcat) x (1 + Kcer/1 + Kce) x R/Kesr
Divide Vmaxapp by Eo
Kme x (1+ R/Ker)/(1+R/Kesr)
Binds to E
Increase - as Ker is large and Kesr is 0 so R/Kesr goes to infinity
Kme x (1+ I/Ki)
Kcat as Kcer = infinity and Kesr = 0
The same
What is the feature of an uncompetitive inhibitor?
What is the Kmapp of a uncompetitive inhibitor going to be and why?
What is the Kmapp equation for an uncompetitive inhibitor?
What is the Kcatapp of an uncompetitive inhibitor going to be and why?
What does this show about Vmaxapp?
Binds to ES
Decrease as Ker = 0
Kme/ 1 + I/Ki
Ker = 0 Kcer = 0 but Kser > 0 so Kcatapp decreases
Vmaxapp decreases
What is the feature of a non-competitive inhibitor?
What is the Kmapp of a non-competitive inhibitor going to be and why?
What is the Kmapp equation for a non-competitive inhibitor?
What is the Kcatapp of a non-competitive inhibitor going to be and why?
What does this show about Vmaxapp?
Binds to both E and ES with the same affinity
No change as Ker = Kesr
Kmapp = Km
Kcer = 0 so Kcatapp increases
Vmaxapp decreases
How do you calculate the number of thiol groups in ovalbumin if the moles of cys = 180nmol, MW of ovalbumin is 45,000 and you have 5mg/ml of ovalbumin in 0.4ml?
What does the position of the cysteine residues in the protein say about its reactivity?
Therefore in what state is the protein most reactive and why?
- 5mgl/ml * 0.4ml = 2mg of ovalbumin
- 45,000 g/mol so 0.045 mg/nmol
- 2mg / 0.045mg/nmol = 44.4nmol ~ 45nmol
- 180nmol/45nmol = ~4 free cysteine residues
Cysteine residues are on the interior of folded proteins, due to little reactivity (as it is folded)
Unfolded as more of the cysteines are accessible by 5,5’-dithio-bis(2-nitrobenzoate)
In the assay for beta galactosidase, what happens on addition of the ONPG substrate?
What is Km & what does it show?
What does a low Km mean?
What does steady state assume in MM?
ONPG is an analogue of lactose & is broken down into O-nitrophenol which appears yellow - goes from colourless to yellow
Substrate concentration at half Vmax so shows the affinity the enzyme has for substrate
Enzyme has high affinity for substrate
Transient where [S] = [S]o while [ES] builds up (burst-phase) to reach the steady state where 1-e^(-at) = 1
