LAB EXAM LAB 2: NADH dilution Flashcards

1
Q

true or false: there are many physiolgy sitautions where chemical events result in production of tandem products

A

true

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2
Q

how come you can take a blood samply and determine the amount of lactate in the blood due to a bout of exercise

A

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

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3
Q

true or false: measuring lactate is useful and cheap

A

false \

it is useful but it can be lengthy and costyl

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4
Q

what is the compound that is produced in tanden with pyruvate and in eeuqal concenttrations to lactate

A

NADH

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5
Q

is lactatet concentrtataion DIRECTLY measured by mreasuring the concentration of NADH

A

indirectly

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6
Q

what is the enxyme that cahnges lactate to pyruvate

A

lactate dehydrogen

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7
Q

what is the reaction of lactate to pyruvate

A

lactaute (c3H6o3) to Pyruvate (c6h4o3) by LADH

While NAD+ is reduced to NADh

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8
Q

how come lactate and pyruvate are simulat

A

lacttae is pyruvae with a pair of hudrrened added on

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9
Q

how can lactate concentration be dtermined

A

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)

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10
Q

what is the equip that allows concentration of lactate to be indirectly measured by measuring concentration of NADh

A

spectrophotometer

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11
Q

true or fasle: epct has the ability to measure concentration of lactate with easy where as it cannot directly measure concentration of NADH

A

false opposite

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12
Q

what is the use of the spect

A

to measure the absorption of light whena. beam passes through a sample

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13
Q

NADH absorbs at what wavelentgh

A

340 nm

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14
Q

do NAD+ and absorb light at 340 nm

A

no

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15
Q

what is the waavelength that the spect is set to

A

340 nm

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16
Q

the amount of light absrobed depends directly on what

A

on the amunt of NADH in the sample (which is proportionate to the amunt of lactae in orginal sample)

17
Q

True or false: NADH and NAD both absorb at 260

18
Q

spect cant only be ised tp assess the lactate/NADH concentration

A

false

it can be used to assess substrate=by product concentration of many different sotaution

19
Q

what is NADH

A

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)

20
Q

what is the role of NADH

A

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

21
Q

what is the difference between FAD and NAD

A

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

22
Q

what is the goal of lab 2

A

Using a spect, generate a NADH standard
curve to determine the concentration of an
unknown sample of NADH.

23
Q

what is a spect

A

Used to ID a solutions’ components by measuring

the amount of light absorbed by that solution.

24
Q

what are 3 examples of things a spect can measure

A

glucose levels
chorlesterol levels
enxyme activites

25
ehat yype of info can the spect provide about the tthings it measures
Healthy/Diseased • Overall metabolic profile • Active/Inactive
26
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. ```
27
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
28
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
29
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
30
how much can the cuvette hold
3000 microliters (3ml)
31
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
32
how to calculate concentrations
divide the concentration of the stock. by the dilution factor !! (EX: 100 mM/4= 25 mM (concnetration of new solution)
33
true or false: you do not need to make replicates of the concentrations
false | important because it controls for pipetting errors
34
once the concentrations diluted are prepared what must you do before placing them in cutte
wrap in parafilm and vortex
35
in order to minimize contamination, what must you do to the cuvette between each concentration
rinse with distilled water
36
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
37
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)
38
measure and note the absorbances of the samples at what wavelength
340
39
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