Practical 1 - Spectrophotometry and Electrophoresis

Question 1

Define electrophoresis

Answer 1

Method of analysing molecules based on charge, by measuring their migration in an electric field.

Question 2

Define spectrophotometry

Answer 2

Method of analysing molecules based on their spectral properties

Question 3

What is absorbance?

Answer 3

Fraction of incident light absorbed by a solution which is measured.

Absorbance = log10 (light transmitted through blank solution/light transmitted through test solution)

Question 4

At which wavelength is there maximum haemoglobin absorption?

Answer 4

540nm. Light is green at this wavelength

Question 5

What does addition of sodium dithionite do to haemoglobin?

Answer 5

Removes oxygen

Question 6

What does oxygen binding grant Hb?

Answer 6

Altered e- resonance, so different absorption spectrum to other proteins

Question 7

Methaemoglobin?

Answer 7

Hb with Fe3+ instead of fe2+, caused by oxidation.

This is what gives dried blood its brown colour.

Question 8

What is the Beer-Lambert Law?

Answer 8

A = E x c x I

Absorbance = Extinction coefficient x concentration x path length.

Basically says that the absorbance of a solution is proportional to the concentration of the absorbing solute and to the distance travelled by light through the sample.

It can be used to determine Hb conc in a sample using spectrophotometry results

Question 9

Which is more negatively charged. HbA or HbS?

Answer 9

HbA is more negative

Question 10

What causes the HbS mutation?

Answer 10

Single change of AA at position 6 in the protein from GLUTAMATE to VALINE (uncharged).

Occurs in beta chain