Acetylcholinesterase Activity And Inhibition Flashcards
Main points to remember
- track units (ie micromole, millimole)
- convert abs (abs/min) to conc (M/min) using Beer Lambert law
COSHH Assessment - Physostigmine
- Hazard
- Who might be harmed
- Measures to control risk
- Toxic
- UG students, Staff, PG demonstrators
- SOP, training, supervision, mop up small spills with blue roll, wear nitrile gloves, wear lab coat at all times, wear safety glasses at all times
Substances used:
- Enzyme
- Substrate
- Inhibitor (+what does it do/ what does it treat)
- Other substances used (+ what do they do)
- Acetylcholinesterase
- Acetylthiocholine iodide 0.075 M
- Physostigmine
- reversible competitive inhibitor
- increases Km, same Vmax, increases Km/Vmax
- high binding strength (low Ki)
- tertiary amine
- penetrates BBB and antagonise muscarinic receptors by inhibiting action of AChE
- used in treatment of glaucoma
- thiocholine (colourless, reacted further with ellman’s reagent)
- Ellman’s reagent (DTMB 0.01M, reacts in 1:1 reaction with free thiol groups to produce yellow anion [extinction coefficient = 14,140M-1cm-1 at 420nm], amount of yellow colour = amount of thiocholine produced = enzyme activity)
- Blank solution (buffer: total volume-subtrate volume, 50microL DTNB, 10microL enzyme)
Michaelis-Menten equation
V0=Vmax ([S]/([S]+Km))
- Vmax (mol/sec)
- Michaelis constant Km (mole/L)
Type of Inhibiton Km Vmax Km/Vmax
Competative x x x
Non-competative x x x
Uncompetative x x x
Type of Inhibiton Km Vmax Km/Vmax
Competative ⬆️ = ⬆️
Non-competative = ⬇️ ⬆️
Uncompetative ⬇️ ⬇️ =
Beer Lambert law: What is it? What does it depend on? Equations?
Light of intensity (I0) enter sample, photons are absorbed, lower intensity (I) then leave
- Amount transmitted (T) = I/I0 - %T = Tx100%
Transmittance falls exponentially with increase in concentration
- Amount of light absorbed (A)=log (1/T)=log (I0/I)
A depends on concentration of sample
- A = εcl
- (only valid if A<1.5 as molecules no longer behave as single molecule in conc sample)
- A = amount of light absorbed
- c = concentration (M)
- ε = molar extinction coefficient (M-1cm-1)
- > amount of light that 1mol of sample will absorb in 1cm cell
Beer Lambert law: assumptions for “A is directly proportional to c”
- incidence light is monochromatic (single wavelength)
- absorbance must lie within specified range in BP (checked against reference)
- each molecule in solution acts as independent absorbing species
- > at high conc, molecules interact + have different ground states and electronic levels
- absorption takes place in homogenous solution
- > not applicable for concentrated samples because precipitation cause scatter of light and no longer absorb
Other factors
- association
- dissociation.
- photodegradation
- solvation
- complexation/ adsorption
- fluorescence (drugs can emit their own light if they are excited)
What is the purpose of this practical?
- determine rate of thiocholine formation when Acetylthiocholine is hydrolysed with enzyme AChE
- determine the activity of this enzyme in presence and absence of fresh and aged Physostigmine
Why are we generating rate data at a range of substrate concentrations?
How can we use this data to understand the effects of the inhibitor?
This will allow us to generate a Lineweaver Burk plot from the group data and therefore assess inhibitors effects on Vmax and Km
Why do you plot product formation against time?
To calculate rate (Vo) under the different conditions in order to prepare a Lineweaver Burk plot
How will we get rate data from this?
Accurate rate data is best taken from Vo (initial rate) as this is the time when we have the most accurate understanding of the concentrations of each component
What are the key chemicals/ reagents you need to use?
- Acetylcholinesterase (enzyme)
- Acetylthiocholine iodide solution (substrate)
- DTNB solution (detection reagent)
- Physostigmine solution (inhibitor)
- Aged Physostigmine solution (inhibitor)
What key equipment do you need?
- accurate pipettes/ tips
- cuvettes
- spectrophotometer
Which operations are critical to getting a good result?
- Getting initial concentrations correct
- Accurate pipetting with correct solutions
- Fast mixing and transfer to spectrophotometer (early data is critical!)
What is being done to reduce error in the result?
absorbance zeroed on blank sample (no substrate) - important as DTNB does have weak absorption at this wavelength
