Spectral characterisation of haemoglobin and its derivatives
Introduction
One the functional proteins in vertebrates are the iron-containing haemoglobin which is one of the components of red blood cells (erythrocytes). Haemoglobin primarily carries oxygen to all body parts as its subunits have binding sites for oxygen molecules. While the globin groups provides binding sites for oxygen, the non-protein group known as the haem group which made up of porphyrin has not oxygen binding sites and has iron binding sites. After binding, haemoglobin becomes oxygenated and is known as oxyhaemoglobin (Costanzo, 2007). Deoxygenation occurs when the oxygen molecules are assimilated into the various organs and tissues including the alveoli leading to the formation of deoxyhaemoglobin. Other gases such as carbon dioxide, nitric oxide and carbon monoxide also have ligands which can bind to the haemoglobin ligands. When bound to these gases and iron, haemoglobin cannot transport oxygen due to the formation of methaemoglobin with iron, carbonmonoxyhaemoglobin with carbon monoxide and carbaminohemoglobin with carbon dioxide. These compounds must first be reduced by reductase enzymes to free up binding sites for oxygen but the process is difficult since such compounds are relatively stable. Failure can lead to diseases such as methaemoglobinemia (Jensen, 2009). Such diseases are manifested in the form of dizzy spells and general fatigue as the body oxygen required for energy production.
Deoxyhemoglobin and oxyhemoglobin absorb light of different wavelength and this is the basis of spectral characterization of haemoglobin and its derivatives. Generally, deoxyhaemoglobin absorb light of lower wavelength estimated to be 660 nm. On other, oxygenated blood tends to absorb light wavelength of 940 nm better.
Experiment purpose
The principal aim of this experiment is characterize and determine the absorption spectra of haemoglobin and its derivatives via spectroscopy by first isolating the blood.
Requirements and methodology
Part A.
- To hypotonic shock, the cells contained in 5 ml of blood containing anticoagulant was lysed by mixing with 40ml distilled water, allowed to rest for 30 minutes followed by a 15 minute centrifugation at 1500g. After removing the supernatant, 9% w/v sodium chloride was added to make the solution isotonic.
- The resulting haemoglobin was divided into two portions and labeled A and B. An aliquot of A was diluted using isotonic saline to 0.8 units at 540 nm and using a spectrometer, its spectral characteristics were measured over 500 to 650 range. This was repeated when a few sodium dithionite were added to the cuvette containing the portion of sample A.
- To a portion of sample A, one drop of potassium ferricyanide was added to make methaemoglobin whose spectral characteristics were measured at 500 to 650 range.
- This procedure was repeated for aliquots of sample B and all the results recorded.
Part B
- To determine the content of the haemoglobin sample, 1 ml of pyridine and 1ml of 0.1M NaOH were added to the sample and thoroughly mixed in the fume cupboard
- Sodium dithionite crystals were added and absorption of the liquid formed measured at 557. Using an absorption coefficient of 34.4 mM-1 cm-1 was used to calculate haemoglobin concentration.
Results and discussions
The results were as follows:
Diagram 1: The spectral characteristics of haemoglobin sample B after adding sodium dithionite crystals
Diagram 2: The spectral characteristics of haemoglobin sample B without sodium dithionite crystals
Diagram 3: The spectral characteristics of haemoglobin sample B with potassium ferricyanide
Table 1: Showing the absorbance of Sample A under different wavelengths
| Sample A | |
| Wavelength | Absorbance |
| 500 | 0.003 |
| 505 | -0.500 |
| 510 | -0.500 |
| 515 | -0.500 |
| 520 | -0.500 |
| 525 | -0.500 |
| 530 | 2.500 |
| 535 | 2.500 |
| 540 | 2.500 |
| 545 | 2.500 |
| 550 | -0.188 |
| 555 | -0.500 |
| 560 | -0.500 |
| 565 | -0.500 |
| 570 | -0.500 |
| 575 | -0.500 |
| 580 | -0.500 |
| 585 | -0.500 |
| 590 | -0.500 |
| 595 | -0.500 |
| 600 | -0.500 |
| 605 | -0.500 |
| 610 | -0.500 |
| 615 | 2.500 |
| 620 | 2.500 |
| 625 | 2.500 |
| 630 | 2.500 |
| 635 | 2.500 |
| 640 | 2.500 |
| 645 | 2.500 |
| 650 | 2.500 |
Pigments including haemoglobins absorb light energy at various wavelengths. The spectrum of haemoglobin and other pigments is an expressional function measured using spectrometer. From the experiment, the change in the wavelength in the two samples was determined by oxygenation and deoxygenation and in the case of addition of potassium ferricyanide or sodium dithionite crystals as seen in the case of Sample B. These chemicals interfere with absorption capability of haemoglobin as they form fairly stable compounds when mixed with haemoblobin.
The measurement of the spectra of haemoglobin is important in diagnosis and treatment of blood poisoning especially when gases such as carbon monoxide and compounds such as iron and cyanide are involved (Jensen, 2009). Diseases such as methaemoglobinemia, carbon monoxide and cyanide can be treated can bet treated by giving the patients supplement oxygen (Salah, Samy & Fadel, 2009). When mixed with methylene blue, oxygen can accelerate the oxidation of these complex compounds that hinder the binding of oxygen to the haemoglobin ligands.
Conclusion
Haemoglobin is a vital protein in the body that ensures that energy required by all the body functions as it transports oxygen throughout the body. The differential spectral characteristics of oxygenated and deoxygenated blood are vital in treatment of diseases such as carbon monoxide poisoning. The presence of such compounds affects the spectral characteristics and this is the basis for diagnosing and treating these diseases.
References
Salah, M., Samy, N. & Fadel, M. (2009). “Methylene blue mediated photodynamic therapy for resistant plaque psoriasis”. J. Drugs Dermatol. 8(1): 42–9.
Jensen, F. B. (2009). “The dual roles of red blood cells in tissue oxygen delivery: oxygen carriers and regulators of local blood flow”. Journal of Experimental Biology (The Company of Biologists) 212 (Pt 21): 3387–3393.
Costanzo, L. S. (2007). Physiology. Hagerstwon, MD: Lippincott Williams & Wilkins.