Effect of low temperature on the absorption spectra of haemoproteins; with observations on the absorption spectrum of oxygen
Keilin, D.; Hartree, E.F.
Nature 164(4163): 254-259
1949
ISSN/ISBN: 0028-0836 PMID: 18139359 Document Number: 12212
(1) Since freezing and further cooling to a very low temperature sharpens and intensifies the absorption bands of certain pigments, spectroscopic examination of such cooled material offers an additional and very valuable method for the study of biologically important pigments in cells, tissues and their aqueous extracts. Such observations can be carried out only with a small dispersion spectroscope. (2) This method has made possible the detection and comparative estimation of cytochrome in cells of low respiratory activity where the pigment has not hitherto been seen. (3) It has revealed the presence of components of cytochrome with an absorption band lying between bands b and c in cells where it would otherwise be invisible. This component (e) appears to be as widely distributed as the other components of cytochrome, forming part of the same catalytic system. (4) It has made possible the direct observation of catalase in perfused haemoglobin-free liver and of peroxidase in strips of horse-radish root. (5) Cooling, by suppressing the ionization of certain substances such as pH indicators, changes or completely suppresses their colour and their absorption bands. (6) When the alkaline forms of methaemoglobin or of peroxidase are cooled, their colours and absorption spectra change to those characteristic of their acid forms. (7) While some coloured substances exhibit sharp. ening and intensification of absorption bands at low temperatures, others are characterized by a change in the spectral pattern or even a suppression of the bands. Cooling of a mixture of substances, therefore, often brings into prominence a component the absorption bands of which were previously masked. (8) Solutions or suspensions of coloured substances in 50 per cent glycerol can be frozen in liquid air to transparent glasses the absorption bands of which show sharpening but very little intensification. If such glasses are warmed until denitrification sets in, there is a rapid and very marked intensification in colour and absorption bands : the latter may become more than fifteen times as intense as the bands of the original uncooled material. On re-cooling the mixture, the intensified bands become sharper and show the fine structure. (9) The changes in absorption spectra referred to above are the results of two independent processes : (a) Sharpening of the bands, often accompanied by their shifting and splitting, which is observed in absence of solvent, in solvents which do not freeze, and in solvents which freeze to transparent glasses. This process requires very low temperatures and depends upon the molecular structure of substances. (b) Marked intensification of bands, which is equivalent to a corresponding increase in the optical depth of the coloured substance and which occurs only in solvents freezing to a crystalline mass. This effect is generally obtained when a light-absorbing substance is associated with microcrystals, or other suitable particles present in the medium, which provide properly distributed multiple reflecting surfaces. (10) Finally, attention has been directed to important literature concerning the sharp absorption bands of oxygen in the visible region of the spectrum, and simple experiments have been devised whereby these bands in gaseous and liquid oxygen can be easily demonstrated and studied with a small dispersion spectroscope.
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