Electrophoresis of Haemoglobin Flashcards
What is absorbance
The fraction of light absorbed by a solution- measured by a spectrophotometer- measures proportion of light transmitted compared to a blank.
A=log10( light transmitted through blank solution)
What does a value of A=1 mean
10% of light is transmitted- 90% is absorbed.
What is used as the blank
Demineralised water.
Is 1.0mg/ml the same as 1.0g/l
yes
How can absorbance be calculated mathematically
A= E( extinction coefficient) x Concentration x Path length
Beer-Lambert Law
What is the extinction coefficient
constant for a particular substance at a particular wavelength
Why is spectrophotometry useful
Most proteins absorb light at a particular wavelength, often UV (proteins and DNA)- useful to measure concentration- also allows the course of the reaction to be followed.
Why do some substances not follow Beer-Lambert law
At high concentrations- some proteins may form dimers with different extinction coefficients.
In which direction does Hb run in electrophoresis
Negative to positive.
What are the differences in mobility of HbS and HbA
HbS has a lower net negative charge, and so travels less distance towards the positive anode.
What is the mutation responsible for sickle cell anaemia
Single point mutation in codon encoding amino acid at position 6 in the beta chain of Hb, and at position 6 in the beta chain of Hb, amino acid changes from glutamate to valine. Glutamate is negatively charged, valine is neutral and hydrophobic, This is significant as position 6 is on the exterior of the protein- making it less soluble. Valine forms hydrophobic interactions with alanine and leucine- clumping the haemoglobin molecules together- intracellular precipitates.
What are the consequences of sickle cell anaemia
More fragile red cells- frequently tear- less oxygen transported to the cells
Due to their irregular shape they can aggregate and block blood vessels- ‘occlusion of blood vessels’ - leading to organ failure- infarction/stroke
