Spectroscopy

Question 1

What are the features of a spectrophotometer ?

Answer 1

1. Measures the fraction of the incident light transmitted through a solution or the amount of light absorbed
2. Transmit light of narrow wavelength ranges

Question 2

Different compounds absorb light at ? and what can be used to distinguish compounds ?

Answer 2

Different wavelengths, and as such a spectrophotometer can be used to distinguish compounds

Question 3

Explain the experiment to work out the optimal NADH absorbance (the wavelength that is most absorbed by NADH) ?

Answer 3

1. Use a UV cuvettes (a reference blank ) containing everything except NADH (or NAD) and zero spectrophotometer at each wavelength as required
   - Used in case other compounds in a solution may absorb (although all other reactants are in fact colourless in this case)
2. Measure the absorbance of the solution containing NADH at a variety of
   wavelengths
3. Repeat for NAD+

Question 4

How do you do the Measurement of the Absorption Spectra of NADH and NAD+ ?

Answer 4

1. You need to find a wavelength where there is a difference in absorbance between substrate and products.
2. Plot a graph of absorbance vs wavelength between 240-400nm for both NADH and NAD+ and calculate difference spectrum.

Question 5

What is the name of the laws of light absorption ?

Answer 5

Beer’s - Lambert’s law

Question 6

These laws use calculations to enable ?

Answer 6

The concentration

of your molecule in a sample to be determined from the absorbance measurements of the spectrophotometer

Question 7

What does Lambert’s Law state ?

Answer 7

“that the proportion of incident light absorbed by a transparent medium is independent of the light intensity”

Question 8

Therefore successive layers of equal thickness will transmit ?

Answer 8

The same proportion of light (intensity decreases by the same proportion for equal lengths travelled)

Question 9

Transmission of light decreases exponentially with ?

Answer 9

Increasing thickness of solution (or concentration)

Question 10

What does Beer’s Law state ?

Answer 10

“that absorbance is proportional to the concentrations of the attenuating speciesin the sample”

Question 11

Therefore absorption shows a linear increase with ?

Answer 11

Concentration and is thus independent of the intensity of the light (within limits!)

Question 12

What does the transmittance measure ?

Answer 12

How much of an incident light (Io) is transmitted

Question 13

What does the absorbance measure ?

Answer 13

How much of an incident light (Io) is absorbed

Question 14

Are Transmission and Absorbance related ?

Answer 14

They are inversely related, more light absorbed, less transmitted.

Question 15

The relationship is not ?

Answer 15

Linear, its logarithmic

Question 16

What is LDH activity ?

Answer 16

A glycolytic enzyme found in all body tissues, including: the muscles, liver, heart, pancreas, kidneys, brain and blood cells

Question 17

Enzyme activities are very high in tissues compared with the serum (ie 500 fold) and therefore ?

Answer 17

Leakage of the enzyme from even a small amount of tissue damage can increase the activity 10-fold

Question 18

Elevations of the serum levels of LDH are associated ?

Answer 18

With a number of different disease states, for example myocardial infarctions, liver disease, anaemia, renal disease and muscular dystrophy.

Question 19

Measurement of the activity in the serum is used ?

Answer 19

Clinically for both diagnosis of disease, and monitoring of the progression of the disease following treatment

Question 20

Glycolysis produces ?

Answer 20

Pyruvate from Glucose

Question 21

Pyruvate enters the ?

Answer 21

TCA cycle or fermentation depending on the oxygen state of the cell

Question 22

In fermentation, pyruvate is?

Answer 22

Reduced to lactate to regenerate NAD+

Question 23

LDH is a hydrogen transfer enzyme which ?

Answer 23

Catalyses the reduction of Pyruvate to L-lactate using NADH as the hydrogen donor

Question 24

CH3COCOOH + NADH + H+ → CH3CHOHCOOH + NAD+

What is this reaction ?

Answer 24

Pyruvate to Lactate.

The reaction is reversible and the reaction equilibrium strongly favours the reduction of pyruvate to lactate

Question 25

You will measure the conversion of NADH to NAD+, a decrease in ?

Answer 25

Light absorbance at the wavelength determined previously