LAB EXAM LAB 2: NADH dilution Flashcards
true or false: there are many physiolgy sitautions where chemical events result in production of tandem products
true
how come you can take a blood samply and determine the amount of lactate in the blood due to a bout of exercise
because when a person is exercsing and the substrattes in the muscle (glucose, glycogen) are being metabolised for ATP for the muscles to contract, there may be a build uo of lactate in the msucle
true or false: measuring lactate is useful and cheap
false \
it is useful but it can be lengthy and costyl
what is the compound that is produced in tanden with pyruvate and in eeuqal concenttrations to lactate
NADH
is lactatet concentrtataion DIRECTLY measured by mreasuring the concentration of NADH
indirectly
what is the enxyme that cahnges lactate to pyruvate
lactate dehydrogen
what is the reaction of lactate to pyruvate
lactaute (c3H6o3) to Pyruvate (c6h4o3) by LADH
While NAD+ is reduced to NADh
how come lactate and pyruvate are simulat
lacttae is pyruvae with a pair of hudrrened added on
how can lactate concentration be dtermined
by transmorning any lactate in the blood back to pyruvate with the help of LDH and NAD+
(if add LDH and NAD+ to blood,, lactate will go to pyruvate)
what is the equip that allows concentration of lactate to be indirectly measured by measuring concentration of NADh
spectrophotometer
true or fasle: epct has the ability to measure concentration of lactate with easy where as it cannot directly measure concentration of NADH
false opposite
what is the use of the spect
to measure the absorption of light whena. beam passes through a sample
NADH absorbs at what wavelentgh
340 nm
do NAD+ and absorb light at 340 nm
no
what is the waavelength that the spect is set to
340 nm
the amount of light absrobed depends directly on what
on the amunt of NADH in the sample (which is proportionate to the amunt of lactae in orginal sample)
True or false: NADH and NAD both absorb at 260
true
spect cant only be ised tp assess the lactate/NADH concentration
false
it can be used to assess substrate=by product concentration of many different sotaution
what is NADH
NADH is short for Nicotinamide Adenine Dinucleotide. It is a derivative of vitamin B
(niacinamide) combined with a ribose (5 carbon sugar), a phosphate group and an adenine
=coenzyme (found in cytosol and mito)
what is the role of NADH
The role of NADH in cellular metabolism is directly linked to the role of NAD+. These
compounds are the most important carriers of the H+ atoms that are released during the breakdown of energetic substrates. This breakdown occurs in order to produce ATP,
which subsequently transports the H+ to the respiratory chain of electrons located in the inner membranes of the mitochondrion. There, the H+ are relinquished to the electron acceptors of the respiratory chain. This process is a major component of aerobic metabolism
what is the difference between FAD and NAD
NAD and FAD are both electron (hydrogen) carriers.
is that FAD is able to accept and carry 2 hydrogen ions, whereas NAD can only accept 1 hydrogen ion.
FAD cannot produce more ATP than NAD since NAD enters the ETC earlier
the more ATP can be produced.
every NADH molecule, 3 ATP
every FADH2 molecule, 2 (ATP
what is the goal of lab 2
Using a spect, generate a NADH standard
curve to determine the concentration of an
unknown sample of NADH.
what is a spect
Used to ID a solutions’ components by measuring
the amount of light absorbed by that solution.
what are 3 examples of things a spect can measure
glucose levels
chorlesterol levels
enxyme activites
ehat yype of info can the spect provide about the tthings it measures
Healthy/Diseased
• Overall metabolic
profile
• Active/Inactive
expkain how to use a micropupette
Select the volume. Set the tip. Press and hold the plunger at the first stop. Place the tip in the liquid. Slowly release the plunger. Pause for a second and then move the tip. Insert the tip into the delivery vessel. Press the plunger to the second stop.
what is the main task of lab exercise 2
to generate a standard curve from the known NADH concentration solution (stock solution) and then use the curve to determine unknown NADH concentrion in the unknown sample
how to generate a standard curve
must gtaph relationship between concetnration of a compound (NADH im this case) and the absorbance of loight incurred by solutions that contrain varrying concentrations of the same comppund must be generated
what is a dilution factor
is a dulition factor of 4 is chosen (Doluted 4 times)
1 PART STOCK IS ADDED TO 3 PARTS DISTILLED WATER
how much can the cuvette hold
3000 microliters (3ml)
how to calculate the volume for 1 part
diveide the total volume by the dilution factor
ex: 3 ml/4 parts
0.75 ml/1 part
how to calculate concentrations
divide the concentration of the stock. by the dilution factor !!
(EX: 100 mM/4= 25 mM (concnetration of new solution)
true or false: you do not need to make replicates of the concentrations
false
important because it controls for pipetting errors
once the concentrations diluted are prepared what must you do before placing them in cutte
wrap in parafilm and vortex
in order to minimize contamination, what must you do to the cuvette between each concentration
rinse with distilled water
before measuring the degree of light absorbed by each of the samples, what must you do to the spect
zero it with a blank sample
how do you zero a spect adn whats the purpose
place duvette full of distilled water into the readig device and reset spect (to negative absorbance incurred by cuvette and water)
measure and note the absorbances of the samples at what wavelength
340
true or false: the hattch must remain open at all times
false do not leave it open at any other times besides when putting samples in cuvette
