3.2 - Haemoglobin (Practical Techniques) Flashcards
1
Q
Electrophoresis
A
- method of analysing molecules by measuring their migration in an electric field
- often use a support (agarose for DNA, polyacrylamide for proteins) which act as a sieve through which the molecules migrate
- Hb practical - cellulose acetate as support, in combination with a buffer solution which means that the Hb molecules are negatively charged and migrate towards the anode, to separate normal Hb (HbA) from sickle cell Hb (HbS) on the basis of their net charge
- results: HbA ran further towards positive electrode than HbS, so you can conclude HbA is more negatively charged - due to a point mutation in an AA of the beta chain (glutamate replaced by valine)
- mixed sample mimicked a Hb sample obtained from a heterozygous individual - shows electrophoresis has power to separate a mixture of proteins which differ by a single amino acid
2
Q
Spectrophotometry
A
- way of analysing molecules on the basis of their spectral properties
- spectrophotometer measures the proportion of light absorbed compared with that of a blank
- absorbance is the fraction of incident light absorbed by a solution, measured by a spectrophotometer
- Hb practical - examine the spectral properties of oxyhaemoglobin and deoxyhaemoglobin by measuring the absorbance of Hb solution over a range of wavelengths
- plot absorption spectra for each, and then you can find the concentration of a mystery solution by reading off the absorbance
- results: oxy-Hb has two peaks, deoxy-Hb has one peak –> spectrophotometry can be used to follow changes in O2 binding by Hb, and is used to check the respiratory status of newborns
3
Q
Oxygen binding properties of haemoglobin
A
- 4 subunits, 2 alpha and 2 beta, which form pockets in which the haem groups bind
- haem groups contain a central ferrous iron atom (Fe2+) to which an oxygen molecule can bind
- binding of oxygen = cooperative - binding of one to a haem group results in conformation of other subunits = increased ability to bind to oxygen (positive cooperative effect - affinity of remaining haem groups for oxygen is increased) - sigmoidal nature of oxygen dissociation curve
- myoglobin has one haem group per molecule and displays greater affinity for oxygen, saturating at lower pO2 values - but lack of cooperativity means it is poor at releasing O2 under the same conditions