Final 8 Flashcards
What assay is used to characterize the enzyme?
Activity Assay/ Enzyme Activity Assay
What is the enzyme in the experiment called? What reaction does it carry our? What is another name for the reaction?
Enzyme is carbonyl reductase
* The enzyme carries out a oxidation reduction reaction
* Also called redox reaction
What does the enzyme activity graph look like? What are the axes? Why does the change occur?
The rate increases then decreases or plateaus due to the taking up of the substrate
What are the Enzyme Activity Assay Components?
- Substrate
- Buffer
- Cofactor - Phosphate buffer
- Present experiment: NAD+
- Enzyme
What are the 5 things assay performance depends on?
- The enzyme concentration
- Ionic strength of the buffer
- PH
- Temperature
- Purity of the enzyme preparation
What are the 4 principles of the CR assay?
- Continuous assay - We will monitor it throughout
- NAD+ - No absorbance at 340nm, absorbance is 0
- NAD is the reduced form- It absorbs maximally at 340nm (lambda max)
- Use these to make the assay possible
What is the activity assay specific to?
The enzyme
Define denature and degrade
Denature: the enzyme is losing its 3 dimensional structure (tertiary structure goes to primary structure)
Degraded: The primary structure of the enzyme is broken.
How is the substrate determined? What are we using?
Some enzymes can digest multiple substrates, others may be specific to one.
* For example, CR is specific to steroid. We will be using cholic acid
What buffer are we using?
Buffer
* We are using sodium pyrophosphate (pH 8.9)
What is the cofactor/coenzyme?
- We are using NAD+
What is the absorbance we are using? Is there color?
Spectrophotometer - UV 340nm
* No color product
What is the general formula of the reaction? What is the formula for this experiment?
The substrate + NAD + Enzyme - product + NADHTH + Enzyme
Present experiment: Colic acid + NAD+ + Enzyme -> CR = product + NAD H +
* The enzyme carries out a reaction but stays the same
What is R on the graph? What does it represent? What is the highest point? Why does it plateaus? What is the S?
R is the rate over time
* the rate the substrate is being reacted by the enzyme
* Reaches a maximum velocity
* Plateau because the substrate finishes or the enzyme is no longer active.
* S is substrate
What are the steps of the experiment?
First in a cuvette place the NAD, Buffer, and Substrate.
* Max 1mL cuvette. Can be used within the UV range.
* Used as a starting point to see activity with and without the enzyme.
Read the cuvette at 340nm
* Use spectrophotometer to measure OD at 340nm.
* To get an initial reading. Should be 0 because there is no enzyme, therefore no absorbance.
* However, sometimes there is background noise and therefore a 0 is not achieved.
* Will read as a zigzag.
Add the enzyme after the first measurement has plateaued.
* Once the enzyme is added, the absorbance increases then plateaus after the substrate is consumed.
What is the obtained graph from the spectrophotometer? What are the axes? What does it look like?
Absorbance over time. The Y axes is Absorbance, the X is time. It first pleateus, then accelerates to a peak, then plateus.
How is the absorbance calculate? What is proportional to?
A= e (absorbtivity constant/ extinction coefficient/molar absorbtivity/molar absorption coefficient) * c (concentration) * l (path length of cuvette (usually 1 cm))
e: How strongly the chemical absorbs light at the specific wavelength.
A=ecl
* l is always 1
Absorbance is proportional to concentration
What does The Beer-Lambert law state?
that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance.
How is delta absorbance calculated?
delta A = (change in OD)/ (change in time)
How is enzyme activity calculated?
Enzyme activity = (deltaA/min) x (Total volume in ml) x (Dilution (if there was any dilution))/ (e )x (Volume of enzyme used (ml))
How is the enzyme activity expressed.
- in units. The amount of substrate (in a unit)/ time (in a unit)
- Ex: in our experiment the substrate is in mMol (micromol) and we are using time as a measurement. Ours would be mMol/ minute. Translates to x micromol of the substrate per minute
What is the definition of a restriction enzyme unit?
Restriction enzyme units: ug (microgram) of DNA at 37 deg, at a specific pH.
What is total volume?
- Total volume is always 1mL.
Why is dilution done? How is the dilution factor caluculated?
sometimes the purified product is so pure the enzyme activity will go too fast.
* Ex: If you take 1mL of enzyme and 9mL of dH20, it is a 10X dilution
What is e?
- e (Epsilon): The molar extinction coefficient/ molar absorptivity of the cofactor at the absorbance.
How many replicates does each sample need? Can 2 readings be the same? What should they be and what should be done with them?
Each sample needs 3 replicates - triplicate
* 2/3 readings should be close. Should take the average of the closest samples.
2 readings cannot be the same.
Do these 5 equations.
1. How many ul (microliter) of 20% sugar solution should be used to make 2mL of 5% sucrose.
* C1V1 = C2V2
2. How to prepare 1L of 1X Tris Borate EDTA Buffer from 10X TBE solution. (Dilution)
3. Prepare 100mL of 40% (w/v) polyethylene glycol. (need molecular weight)
4. How to prepare 200mL of 95% (v/v) solution of ethanol. (just multiply)
5. How would you prepare 50mL of 20mm NaOH2. (39.997 g/mol)
What are the two issues with leaky expression?
Can be a waste of energy and create a metabolic burden because there may not be enough protein.
If the protein is toxic, the leaky expression can create proteins that can kill the bacteria prior to induction
How can leaky expression be controlled? How is it produced?
What does it do?
Can control the leaky expression through a lysozyme.
The lysozyme will be produced by another gene and will bind to the RNA polymerase and repress it, controlling the leaky expression.
What can be used in the host to get the genes to control leaky expression? How are there expression? What host can be used and what does it have?
Can use alternative plasma with lyse genes
Lyse genes are constitutively expressed
Can also use BL21DE3 PLYSS/E host
* Has the lysozyme plasmid in it
When will leaky expression only occur?
Leaky expression will only occur when induction does not occur because there is an excess of RNA polymerase.
