Haemoglobin Practical

Question 1

What is electrophoresis?

Answer 1

Method of analysing molecules by measuring their migration in an electric field. We often use some sort of support (e.g. agarose for DNA, polyacrylamide for proteins) which acts as a sieve through which the molecules migrate.

Question 2

What is spectrophotometry?

Answer 2

Way of analysing molecules on the basis of their spectral properties

Question 3

What is the function of RBCs?

Answer 3

Carry oxygen from lungs to tissues.
Transfer CO2 from tissues to lungs.
(Each contains Hb)

Question 4

Where can we find Hb?

Answer 4

Exclusively in RBCs.

Question 5

How much Fe does each gram of Hb contain?

Answer 5

3.4mg

Question 6

Normal Hb concentration in adults?

Answer 6

120-165 g/L

Question 7

In what other proteins can the haem group be found?

Answer 7

Myoglobin, cytochromes, peroxides, catalase

Question 8

Describe the structure of 1 haem group.

Answer 8

Combination of porphyrin ring with central iron atom

(Iron usually in Fe2+ form and is able to combine reversibly with oxygen).

Question 9

List the functional globin chains.

Answer 9

Alpha cluster - alpha and zeta globin genes

Beta cluster - beta, gamma, delta and epsilon genes

Question 10

How many functional globin chains are there?

Answer 10

8 split into alpha (3) and beta (5) clusters

Question 11

Structure of HbA

Answer 11

2 alpha and 2 beta globins (96-98%)

Question 12

Structure of HbA2

Answer 12

2 alpha and 2 delta globins (1.5-3.2%)

Question 13

Structure of HbF

Answer 13

2 alpha and 2 gamma globins (0.5-0.8%)

Question 14

What type of Hb is the vast majority in a healthy adult?

Answer 14

HbA

Question 15

Describe the secondary and quaternary structure of HbA.

Answer 15

Secondary - 75% of the alpha and beta chains are in the form of alpha helices.

Quaternary:
Approximate sphere.
Hydrophilic surface (charged polar side), hydrophobic core. Haem groups found within pockets.

Question 16

Safety protocols for electrophoresis and spectrophotometry.

Answer 16

Human blood therefore always wear gloves.
All sharps go in appropriate sharps bins (yellow)
No eating/drinking
Lab coats/aprons worn at all times.
Use blue trays to pass equipment between yourselves.

Question 17

What one word describes the binding of oxygen to Hb and explain what this means?

Answer 17

COOPERATIVE
Binding o​f 1 O2 molecule to a haem group results in a change in the conformation of the other subunits, affecting their ability to bind oxygen. In Hb, this is a positive cooperative effect, meaning that as oxygen binds to one haem group, the affinity of the remaining haem groups for oxygen is increased. This is exemplified by the sigmoidal nature of the oxygen association curve for haemoglobin shown below.

Question 18

In which organ is Hb oxygenated?

Answer 18

Lungs

Question 19

Is there a higher pO2 in the lungs or tissues surrounding by circulation outside of lungs?

Answer 19

Lungs (100 torr)

Elsewhere pO2 is ~20 torr

Question 20

How does the positive cooperatively displayed by the binding of O2 effect the amount of O2 that can be taken up by Hb?

Answer 20

Almost 2x as much oxygen than would be possible in the absence of cooperativity.

Question 21

What molecule has a higher affinity for oxygen than Hb and why?

Answer 21

Myoglobin
Saturates at lower pO2.
However, the lack of cooperativity means that it is poor at releasing oxygen under the same conditions.

Question 22

Where is myoglobin (protein) found?

Answer 22

Muscle

Question 23

How many haem groups per molecule can myoglobin bind?

Answer 23

1 haem per molecule

Question 24

Where are the 2 main peaks for oxyHb found on a spectrophotometry curve (absorbance against wavelength)?
Compare this with the peaks in the results for deoxyHb.

Answer 24

540 and 580 nm (plus one at 412nm)

In the deoxyHb, the spectrum changes, with a single peak in this range at 560 nm (plus one at 430 nm).

Question 25

What colour difference would you see between oxyHb and deoxyHb?

Answer 25

Deoxy-Hb having a blueish-purple tinge compared to oxy-Hb. If insufficient dithionite was added then the spectrum are likely to have overlaid each other.

Question 26

Why is spectrophotometry medically relevant?

Answer 26

Used when checking the respiratory status of newborn infants. The spectrum also changes for other forms of Hb, which might need to be examined for diagnostically.

Question 27

What is a pulse oximeter?

Answer 27

Non-invasive way of measuring oxygen saturation levels. Relies on the difference in absorbance of oxyHb and deoxyHb. The oximeter is clipped onto a thin appendage (finger, ear lobe) and the device emits light at two wavelengths, 660nm (red light) and 940nm (infrared light) via a couple of LEDs.

OxyHb absorbs more infrared light but less red light than deoxyHb. 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.

Question 28

How is carboxyHb generated?

Answer 28

Binding of CO to ferrous iron (Fe2+) in haemoglobin.

Question 29

Does Hb have a higher affinity for CO or O2?

Answer 29

Hb has a 200-fold greater affinity for CO than O2, therefore it can readily outcompete O2 for binding to the four haem groups of haemoglobin. Even relatively low levels of CO can dangerous. Levels of 0.2% CO can lead to death within an hour or two.

Question 30

How is methaemoglobin (MetHb) generated?

Answer 30

Generated when the Fe2+ ion is oxidised to the Fe3+ (ferric) state which results in greatly impaired oxygen binding. This gives the blood a bluish/chocolate colour if present at high levels.

Question 31

How do relatively low levels of MetHb affect the oxygen-dissociation curve?

Answer 31

Shift leftwards, which can result in tissue anoxia, as oxygen is not readily released by MetHb.

Question 32

What enzyme reduces MetHb to Hb?

Answer 32

Methemoglobin reductase

Question 33

How can methaemaglobinaemia happen?

Answer 33

Can be hereditary, e.g. deficiency in methemoglobin reductase or production of a mutant form of Hb known as Hb M, which is resistant to reduction.

Methaemaglobinaemia can also be acquired following exposure to chemicals including aniline residues such as p-chloroaniline, nitrates, and local anaesthetics such as benzocaine.