Haemoglobin Flashcards
What protein is involved in the transport of oxygen from the lungs to the tissues?
Haemoglobin
What can you use to determine concentration of something, and why?
spectrophotometer
The absorbance of a solution is proportional to the concentration of the absorbing material within it and to the distance (or path length) travelled by the light through the sample.
What is the Beer-Lamber law?
A= E x c x L
absorbance of the solution
concentration
path length
extinction coefficient
What is the extinction coefficient?
a constant for the substance being measured at that particular wavelength
What are the 2 main functions of haemoglobin?
to efficiently transport oxygen from the lungs to the tissues
help transport CO2 back to the lung
Describe the structure of haemoglobin?
- Consists of 4 globin chains, HbA has 2 alpha chains and 2 Beta chains
- Each globin chain contains a haem group
- Each Haem group is made up from a central ferrous iron atom (fe2+) surrounded by a porphyrin ring
How is the binding of oxygen to haemoglobin described?
cooperative
- meaning that binding of one O2 molecule to a haem group results in a change in the conformation of other subunits, affecting their ability to bind O2
What type of effect is there in haemoglobin?
Positive cooperative effect
- meaning that as oxygen binds to one haem group, the affinity of the remaining haem groups for oxygen is increased
How is the positive cooperative effect shown?
Exemplified by the sigmoidal nature of the oxygen association curve for haemoglobin
Where in humans is the partial pressure of O2 approaching 100 torr, and the haemoglobin is almost completely saturated with oxygen (98%)?
The lungs
Why might some substances fail to obey the beer-lambert law?
for example, at high concentrations a protein might form dimers with a different extinction coefficient)
For hypothetical noncooperative oxygen transport protein, the maximum amount of oxygen that can be transported from regions with identical pO2 values is what, and what does it show?
38%
- shows that, positive cooperativity displayed by the binding of oxygen to haemoglobin enables it to deliver almost twice as much oxygen than would be possible in the absence of cooperativity
What is myoglobin?
an oxygen binding protein found in muscle, has one haem group per molecule and displays greater affinity for oxygen than haemoglobin
saturating at lower pO2 values
the lack of cooperativity means that it is poor at releasing oxygen under the same conditions
What four chains are HbA molecules made up from?
Two alpha chains and 2 beta chains
What two chain type is fetal haemoglobin made from?
Two alpha chains and two gamma chains
What is meant by the positive cooperative binding which oxygen displays?
This means that it becomes easier for oxygen to bind as more oxygen binds to it - hence resulting in the sigmoidal curve which arises when oxygen dissociation is plotted
How is the oxygen affinity different in fetal haemoglobin compared with adult haemglobin?
The oxygen affinity is higher
How does HbF have a higher oxygen affinity than HbA?
A single AA substitution results in reduced affinity for 2,3-DPG and therefore a higher oxygen affinity
Why is it essential that fetal haemaglobin has a high affinity for oxygen?
This means at very low concentrations of oxygen, which the placenta has, HbF can still readily bind the oxygen which the HbA is offloading
Why can myoglobin not display positive cooperativity?
It only has one oxygen binding site
What is the p50 value for myoglobin and haemaglobin respectively?
Myoglobin = 2
Hb = 26
Where is myoglobin found?
Predominantly in the muscle cells
Why is myoglobin useful?
It is a store of oxygen, which is mainly found in the muscles, and has a high affinity fr oxygen - it only releases the oxygen it is storing when the oxygen concentration is very low
A right shift in the oxygen dissociation curve is associated with what change?
A decrease in the oxygen affinity
A left shift in the oxygen dissociation curve is associated with what change?
An increase in the oxygen affinity
What factors could cause an increase in the oxygen affinity (Left shirt)?
Decreased CO2, Decreased 2,3-DPG, Decreased H+
What factors could cause a decrease in the oxygen affinity (right shift)?
Increased CO2, Increased H+, Increased 2,3-DPG and increased temperature
Describe how a left shift in the oxygen dissociation curve results in an increase in the affinity for oxygen?
When the curve shifts left, at the same partial pressure of oxygen, the saturation of the haemoglobin molecules is higher. This is because as the affinity is higher, the Hb is less willing to release the oxygen molecules and therefore the Hb is more saturated
How is 2,3-DPG produced?
Rapoport-Leubering Shuttle
How does 2,3-DPG affect Hb?
it stabilises the structure and reduces the affinity for oxygen
what is the p50 value?
The value at which 50% of the oxygen molecules have bound
Why is Hb described as a tetramer?
because it has 4 globin chains
Describe what is meant by globin gene switching, and when does it occur?
Occurs at birth - this is the switch from gamma to beta chains, resulting in HbA not HbF
What is Carboxyhaemaglobin?
Molecule formed when carbon monoxide binds to Fe2+ (ferrous iron) in the haemoglobin
How much greater is haemoglobin’s affinity for CO than O2?
200-fold greater affinity
- therefore it can readily outcompete oxygen for binding to the four haem groups of haemoglobin
What level of CO can lead to death in an hour or two?
0.2%
what is MetHb?
Methaemoglobin
How is MetHb generated?
Formed when Fe2+ ion is oxidised to the ferric state (Fe3+)
What does the oxidation of Fe2+ to Fe3+ in Methaemoglobin lead to?
results in greatly impaired oxygen binding. this gives the blood a bluish/ chocolate colour if present at high levels.
What can relatively low levels of methaemoglobin (MetHb) lead to?
it can cause the oxygen dissociation curve to shift leftwards, which can result in tissue anoxia, as oxygen is not readily released by MetHb
What is present if your blood has a blueish/chocolate colour?
Methaemoglobin
What enzyme is used to reduce methaemaglobin back to haemaglobin?
methaemoglobin reductase
What affect does MetHb have on oxygen?
MetHb can carry oxygen, however cannot release it to the tissues
What is Methaemaglobinaemia?
disorder where…
there is a deficiency in methemoglobin reductase
or
production of a mutant form of haemoglobin known as haemoblin M- which is resistant to reduction.
How can Methaemoglobinaemia be acquired?
Hereditary
Exposure to chemicals like aniline dyes (e.g., p-chloroaniline), nitrates, benzene and local anaesthetics like benzocaine
What does the absorbance spectrum look like for oxy-haemoglobin?
There are two peaks, with one at 540 and 580nm
(if the investigation had been extended, you may have also found a peak at 412nm)
What does the investigation and beer-lamberts law show?
absorbance is linearly proportional to concentration
Why does the spectrophotometer eventually become non-linear?
as absorbance gets too high
- but it should always be good up to an absorbance of 1.0
- the best fit straight line should be drawn to the experimental points
What is the ideal wavelength to set the haemoglobin absorbance spectrum at?
540nm
How many peaks does deoxy-Hb have and where are they/it?
1 at 560nm (plus on eat 430nm)
What is the colour of deoxy-Hb?
Blueish- purple tinge compared to oxy-Hb
What could’ve happened if insufficient dithionite was added?
the spectrum are likely to have overlaid each other
When is the spectrophotometry used clinically?
To check the respiratory status of newborn babies
What is pulse oximetry?
A pulse oximeter is a non-invasive way of measuring oxygen saturation levels
What does pulse oximetry rely on?
It relies on the difference in absorbance of oxyhemoglobin and deoxyhaemoglobin
How does the pulse oximetry work, e.g., where is it placed etc.?
It is clipped onto a thin appendage (finger, ear lobe) and the device emits light at 2 wavelengths
660nm (red light) and 940nm (infrared light) via a couple of LEDs
What absorbs more red/ infrared light- oxy-Hb or deoxy-Hb?
Oxygenated Hb absorbs more infrared
deoxygenated Hb absorbs moer red
How are readings taken from pulse oximetry?
The transmitted light at each wavelength is corrected for the pulse of arterial blood by subtraction of the minimum absorbance.
The ratio of absorbances at each wavelength can be converted to SpO2 by reference to the Beer-Lambert law
Which is more negative HbA or HbS?
HbA is more negative
Why is HbS not as negatively charged as HbA?
A single point mutation occurs in the Beta chain of HbS - glutamate is replaced by valine
What affect does the single base substitution which occurs for HbS to form, have?
The substitution from Glutamate to Valine is one from
hydrophilic and negatively charged to hydrophobic and positively charged
In gel electrohoresis, how can HbS be differentiated from HbA?
HbS is sickled Hb - this occurs due to a point mutation which is glutamate is replaced by valine (negative to positive).
Therefore, the HbS will be less negative and therefore not move as far towards the positive electrode as HbA will
Why do foetal red blood cells produce haemoglobin F rather than haemoglobin A?
Haemoglobin F has a higher affinity for oxygen than haemoglobin A which facilitates transfer of oxygen from the mother to the foetus.
How does chromatography on cellulose separate proteins?
on the basis of their charge
What is the role of the Rapoport–Luebering shuttle in red blood cells?
To generate 2,3-BPG which is important for the cooperative binding of oxygen to haemoglobin A.
In electrophoresis of does HbA or HbS move further?
HbA moves further, towards the positive electrode (bottom of gel)
therefore it is more negatively charged
What causes the difference in charge of HbA and HbS?
Due to a point mutation occurring in one AA of the beta chain
Glutamate in the normal protein (hydrophilic and negatively charged) is replaced by valine (hydrophobic, uncharged)
What were the three forms of haemoglobin used in the electrophoresis?
HbA, HbS and mixture of HbA & HbS
