268 



PORPHYRINS 



620 600 580 560 540 



a, 

 603 



564 



c 

 550 



i 



50 0m>M 



519 



FIGURE 13-1 



The bands labelled a, b, and c represent the so-called a-bands of cytochromes a, b, and 

 c. Band d represents the combined i3-bands of cytochromes b and c. If bands are not 

 observable in the intact tissue because other pigments interfere (e.g. chlorophyll in 

 leaves) or because the hematin compounds are too dilute, modified approaches may be 

 possible (20). Interfering pigments can be removed by solvent extraction and/or a par- 

 ticulate preparation from the homogenized tissue used for observation as in the procedure 

 of Bhagvat and Hill (21). Faint bands may be intensified by observation at the temperature 

 of liquid air. This intensification, which may be as much as 20-fold, depends on the mi- 

 crocrystalline structure of the frozen medium rather than any change in the pigments 

 themselves. A sensitive method for determining total hematin involves treatment of the 

 material with pyridine (20%) and sodium dithionite (1%) in alkaline solution (0. IN NaOH). 

 This results in denaturation of associated proteins and formation of hemochromogens 

 from any iron porphyrins which may be present. In these derivatives the 5 and 6 coordi- 

 nation positions of ferrous porphyrin are occupied by pyridine, and a strong absorption 

 band at 556 m/i results for protoporphyrin hemochromogen, at 551 m^. for the hemochro- 

 mogen from cytochrome c, and 585 mfx for the hemochromogen of the A cytochromes. 

 Hemochromogen formation may be made the basis for quantitative estimation of total 

 hematin in plant tissues (22). 



The absorption spectra of chlorophyll derivatives may be observed in intact tissues 

 using the direct vision spectroscope, but more frequently purified preparations are 

 studied spectrophotometrically. The exact positions of absorption maxima may vary 

 slightly with the solvent used. Figure 2 gives the spectra for chlorophylls a and b. The 

 sharp band at about 660 mji is characteristic of dihydroporphyrins. It is also seen in 

 pheophytin, but the addition of chelated magnesium makes it even more intense. The ab- 

 sorption spectroscopy of chlorophyll and other porphyrins has been reviewed by Rabino- 

 witch (23) and Aronoff (24). The application of spectrophotometry to quantitative meas- 

 urement of chlorophyll is described by Smith and Benitez (17) and Bruinsma (25). 



Fluorescence spectra of porphyrins have also been extensively studied and are im- 

 portant in characterization. However, among natural products only the metal-free por- 

 phyrins and chlorophylls fluoresce. The iron porphyrins do not. Fluorescence spectrum 

 curves for a variety of compounds are presented by French et al. (26). 



The second most important property in characterizing porphyrins has been their 

 solubility behavior especially with regard to dilute hydrochloric acid. This solubility is 

 naturally related to the proportion of polar and non-polar groups in the molecule. Phytol- 

 containing compounds are much less soluble than compounds with free carboxyl groups, 

 so that partition between ether and various concentrations of HCl has been useful in sepa- 

 rating porphyrins from mixtures. Quantitatively a "hydrochloric acid number" can be 

 assigned to each porphyrin and defined as the percentage concentration of HCl which will 

 extract 2/3 of the compound from an equal volume of its ether solution (usually at a con- 

 centration of 0. 02%). Some representative HCl-numbers are given in the accompanying 

 table on p. 270. 



