THE OPTICAL ABSORPTION SPECTRUM OF HAEMOGLOBIN

Work as one group per spectrophotometer for this exercise, while waiting for 1.5 hours for the ammonia assay colour development

Introduction

One of the most important functions of the blood is the transport of oxygen and carbon dioxide to and from the tissues. The solubility of oxygen is low, but the carrying capacity of the blood is increased by molecules that bind reversibly with oxygen, taking it up at the respiratory surfaces and releasing it to the tissues. These molecules are proteins with metal ions, which give them complex patterns of optical absorbance. They are thus coloured, and termed respiratory pigments.

The optical absorbance of the pigments differs between the oxygenated and deoxygenated states. This is of no functional significance to an animal, but is of use to the physiologist. If the absorbance of the pigment is known in both fully oxygenated and fully deoxygenated states, then the change in absorbance can be used as a measure of the percentage oxygenation of the blood. This information is used to construct oxygen dissociation curves, showing the oxygenation at different partial pressures. In this exercise you will measure the absorption spectra of mammalian haemoglobin in both the oxygenated and deoxygenated states.

Procedure

  1. Place 0.1 ml of blood (you will be given information on the species used) in a boiling tube and dilute to 15 ml with distilled water. This disrupts the erythrocyte membranes and releases the haemoglobin into solution.

  2. Ensure that the haemoglobin is fully oxygenated by bubbling compressed air slowly through it for 30 s.

  3. Set the wavelength of a spectrophotometer to 540 nm. Switch on the machine. Set the Zero Transmittance (if required).

  4. Place 2 ml distilled water into a cuvette as a "blank". Open the sample compartment and insert the cuvette containing the blank into the sample compartment.

  5. Note the zero absorbance or zero the machine if this is possible.

  6. Place 2 ml of the oxygenated diluted blood into another cuvette. Remove the blank cuvette and replace with the sample cuvette. Close the lid and read the absorbance. Call a demonstrator to check whether your sample needs to be diluted before further measurements.

  7. Measure absorbance over the range 450-600 nm against the distilled water blank. Change the wavelength in 10 nm steps, but use 5 nm intervals in regions where there are sharp peaks or troughs. Check the zero transmittance and reset the zero absorbance using the blank at each wavelength.

  8. Add a small amount (pre-weighted) of the reducing agent sodium dithionite (Na2S2O4.H2O) to the sample in the cuvette to chemically deoxygenate the haemoglobin. Add the same amount of sodium dithionite to the blank. Cover each cuvette with parafilm and invert to mix, but do not shake. Allow to stand for 5 min.

  9. Repeat the measurement of the absorption spectrum from 450-600 nm, for deoxygenated haemoglobin. Use the blank with sodium dithionite, checking the zero transmittance and resetting the zero absorbance at each wavelength

  10. Plot the absorbance against wavelength to give a graph of the absorption spectra of both oxyhaemoglobin and deoxyhaemoglobin, on the same axes.


Writing your report

Present a graph of the absorption spectra of oxyhaemoglobin and deoxyhaemoglobin of the named mammal. How do they compare with absorption spectra of the respiratory pigments of other animals, and why ?