Staniforth

Question 1

what is the difference between a direct and a quenching method

Answer 1

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

Question 2

what does non-invasive mean, what methods are non invasive

Answer 2

does not effect the course of the reaction, direct

Question 3

what are disadvantages of direct methods

Answer 3

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

Question 4

what is an advantage of direct measurement

Answer 4

it is real time?
fewer steps means smaller errors and better signal to noise
quicker

Question 5

what is an advantage of quenching methods

Answer 5

can be used for reactions which cannot be measured directly
can measure all components

Question 6

what are the 3 steps of quenching methods

Answer 6

triggering: starting the reaction
quenching: stopping the reaction - done by a quenching agent
^ done with the same instruments
quantification/identification

Question 7

factors to consider when choosing an assay

Answer 7

range of applicability
timescale
sensitivity
availability
accuracy
cost

Question 8

what timescale reaction would allow you to manually mix the reactants

Answer 8

impossible < 10 seconds - 1 minute < accurate

Question 9

what is equilibrium perturbation

Answer 9

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

Question 10

when in doubt

Answer 10

hplc

Question 11

why would you choose radioactivity as the signal

Answer 11

when sensitivity is required —conc?

Question 12

what is fluorescence

Answer 12

a property of some compounds where light is absorbed at one frequency and emitted at another

Question 13

how is fluorescence measured

Answer 13

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

Question 14

how can pH change in reaction be measured

Answer 14

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

Question 15

what is SPR: surface plasmon resonance

Answer 15

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

Question 16

what are the disadvantages of SPR: surface plasmon resonance

Answer 16

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

Question 17

what is the dead time of apparatus

Answer 17

time before you’re able to measure a reaction

Question 18

what is the Io (o in subscript) in absorbance

Answer 18

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) )

Question 19

what is I in absorbance

Answer 19

the transmitted light which leaves the sample towards to photomultiplier (detector)
subtract from the Io to get the absorbance (beers law = log(lo/I) )

Question 20

what does a photomultiplier do

Answer 20

transforms observed photons into an electrical signal

Question 21

what tells you that the measured signal of absorbance is directly proportional to the concentration of the absorbing compound (equation)

Answer 21

A = e.c.l (e = eta)

Question 22

what is the refractive index

Answer 22

measurement of a property (of all compounds) to reflect light, highly sensitive and dependent on mass bound
used by SPR

Question 23

what happens to the solutions in continuous flow

Answer 23

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

Question 24

what is the detector in continuous flow

Answer 24

CCD detector - like a camera, resolves light coming out in space
resolves the length of the observation cell for timepoints

Question 25

what qualities of compounds make them suitable for use in stopped flow

Answer 25

fluorescence or absorbance

Question 26

describe the set up of a stopped flow device

Answer 26

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

Question 27

what would you use to start and stop a reaction when the reaction timescale is shorter than a minute

Answer 27

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

Question 28

how would you measure radioactivity once quenched and components separated

Answer 28

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

Question 29

what does hplc stand for

Answer 29

high performance liquid chromatography

Question 30

how does hplc work

Answer 30

column chromatography, stronger column matrices, higher pressure and automated injection
optical or fluorescence signal

Question 31

starting a timer

Answer 31

with other hand to the pipette

Question 32

what is the output of hplc

Answer 32

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

Question 33

quenching agent acid - precaution

Answer 33

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

Question 34

precautions with a coupled assay

Answer 34

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