Staniforth Flashcards
what is the difference between a direct and a quenching method
in direct methods the reaction is observed directly as it happens - non invasive so do not affect the course of the reaction
in quenching the reaction is arrested and the extent of the reaction assessed at each timepoint - usually uses aliquots
what does non-invasive mean, what methods are non invasive
does not effect the course of the reaction, direct
what are disadvantages of direct methods
need a probes which is specific to the chemical of interest so it is hard to monitor all components of a reaction simultaneously
type of reactions that can be measured directly is limited
what is an advantage of direct measurement
it is real time?
fewer steps means smaller errors and better signal to noise
quicker
what is an advantage of quenching methods
can be used for reactions which cannot be measured directly
can measure all components
what are the 3 steps of quenching methods
triggering: starting the reaction
quenching: stopping the reaction - done by a quenching agent
^ done with the same instruments
quantification/identification
factors to consider when choosing an assay
range of applicability
timescale
sensitivity
availability
accuracy
cost
what timescale reaction would allow you to manually mix the reactants
impossible < 10 seconds - 1 minute < accurate
what is equilibrium perturbation
they do not mix the 2 solutions but rely on other factors to start the reaction eg. laser, changes in temp/pressure
called equilibrium perturbation because these changes effect the position of equilibrium
observe the reaction relax to equilibrium
when in doubt
hplc
why would you choose radioactivity as the signal
when sensitivity is required —conc?
what is fluorescence
a property of some compounds where light is absorbed at one frequency and emitted at another
how is fluorescence measured
same as in a spectrophotometer light emitted from a lamp is passed through a monochromator which excited the sample at the specific frequency at a 90 degree angle there is another monochromator which selects for a specific frequency from the light emitted from the sample which goes to the photo amplifier in the detector
quantitative
how can pH change in reaction be measured
with a pH-stat reaction vessel has a pH meter on an electrode, a stirrer and a pH sensitive pump to ass NaOH or HCl to maintain the pH in the reaction vessel
read out is how much acid/base is added, tells you how much of the other is produced
what is SPR: surface plasmon resonance
does not require the reaction to have an optical signal, determines the Kd (dissociated constant for a ligand binding to a protein
L + P <–> LP
where <- is Kon and -> k off
kd = koff/kon
ligand flows across a surface, change measured in refractive index, light bounded off
what are the disadvantages of SPR: surface plasmon resonance
its descriptions of relative binding of different compounds is accurate but it is hard to extrapolate the value to bulk solution as it is not a perfect mimic
but good at suggesting what drugs bind the tightest
what is the dead time of apparatus
time before you’re able to measure a reaction
what is the Io (o in subscript) in absorbance
the incident light - the light which is produced by the monochromator (selected for wavelength), towards the sample (cuvette or observation cell)
minus the to get the absorbance (beers law = log(lo/I) )
what is I in absorbance
the transmitted light which leaves the sample towards to photomultiplier (detector)
subtract from the Io to get the absorbance (beers law = log(lo/I) )
what does a photomultiplier do
transforms observed photons into an electrical signal
what tells you that the measured signal of absorbance is directly proportional to the concentration of the absorbing compound (equation)
A = e.c.l (e = eta)
what is the refractive index
measurement of a property (of all compounds) to reflect light, highly sensitive and dependent on mass bound
used by SPR
what happens to the solutions in continuous flow
push 2 liquids into a small capillary tube using a pneumatically (pressure driven ram) which goes at a constant rate
a platinum ball where the two tubes meet works as a mixed
what is the detector in continuous flow
CCD detector - like a camera, resolves light coming out in space
resolves the length of the observation cell for timepoints
what qualities of compounds make them suitable for use in stopped flow
fluorescence or absorbance
describe the set up of a stopped flow device
syringes of enzyme and substrate driven by a pneumatic ram –> go into an observation cell which has a lamp
a stopping syringe also goes into the observation cell which is operated by a microswitch and computer
what would you use to start and stop a reaction when the reaction timescale is shorter than a minute
quenching version of stopped flow enzyme and substrate pushed together in a syringe –> aging loop
a computer determines the 2nd push of H2O (or other buffer) to the start of the aging loop and the quenching gent to the end where it is collected in the collection tube
- dead time: 10ms
how would you measure radioactivity once quenched and components separated
a scintillation counter detects radioactivity in counts per minute (cpm)
a chemical - scintillant - is added to the sample, converts radiation energy of the radioactive species into light quanta - measured using the standard photomultiplier in the scintillation counter
what does hplc stand for
high performance liquid chromatography
how does hplc work
column chromatography, stronger column matrices, higher pressure and automated injection
optical or fluorescence signal
starting a timer
with other hand to the pipette
what is the output of hplc
a chromatogram where the area of each peak - measure of the amount of each constituent
the peaks may not be able to be identified - further analysis with identification techniques
quenching agent acid - precaution
corrosive
eyewear, gloves etc.
degradation of ATP could occur if left in acidic conditions for too long
precautions also include how to make the assay the most accurate
precautions with a coupled assay
make sure right amounts of all the
different reagents so that you ensure that the reporter reaction is effectively telling you about the
reaction of interest
make sure the reaction of interest
(reaction 1) occurs at a much slower rate than the reaction it is coupled to (reaction 2). In
order to make the assumption that the rate of NADH consumption reflects the production of
pyruvate in reaction 1
