PORPHYRINS 267 



COMPONENT SOURCE AUTHOR AND REFERENCE 



Cytochrome c wheat germ Goddard (10), Hagihara ei aZ. (11) 



Cytochrome f parsley Davenport and Hill (12) 



Peroxidase horseradish Kenten and Mann (13) 



Catalase spinach Galston, Bonnichsen and Arnon (14) 



Hemoglobin soybean nodules Sternberg and Virtanen (15) 



This situation is somewhat better with regard to the chlorophyll pigments. Here 

 the problem is not so much one of obtaining a goodly quantity of pigment as in being as- 

 sured that the pigment obtained is not an artifact, since degradation easily occurs during 

 the purification procedure. Acetone is most commonly used to extract the pigments from 

 either fresh or dried leaves (8(ffc acetone is used for dried leaves). Ethanol is effective, 

 but the enzyme chlorophyllase present in fresh leaves catalyzes the reaction: 



ethanol + chlorophyll -^ phytol + ethyl chlorophyllide 



so that unless chlorophyllase is inactivated or extraction is carried out for a very short 

 time, the chlorophyll will be contaminated. Another pitfall in the initial extraction is 

 the ready removal of magnesium from chlorophyll in acidic solutions. Many plant ex- 

 tracts contain enough organic acids to bring about this degradation to pheophytin. It can 

 be avoided by grinding with acetone in the presence of a weak base such as 1% magnesium 

 carbonate or dimethylaniline. Other precautions may sometimes be taken such as grind- 

 ing in the cold and/or in the dark. 



Some separation of the pigments in the acetone extract may be achieved by solvent 

 partition methods, but column chromatography is generally the method of choice. Water 

 and petroleum ether are added to the acetone extract and the petroleum ether layer which 

 now contains the pigments is washed with water to remove acetone and dried with anhy- 

 drous sodium sulfate before chromatography. The most valuable adsorbent is powdered 

 sucrose. Stronger adsorbents, such as magnesium oxide, which are useful for carotenes, 

 may cause subtle, isomeric changes in the delicate chlorophyll pigments. Anderson and 

 Calvin (16) have found that the purest chlorophyll can be obtained by chromatographing 

 first on powdered polyethylene and then on sucrose. More detailed purification procedures 

 and tests for impurities are given in the review of Smith and Benitez (17) and the book by 

 Strain (18), 



CHARACTERIZA TION 



The observation of absorption spectra is probably the one most important technique 

 for characterization of both hematin and chlorophyll derivatives, although it does not per- 

 mit distinction between closely related compounds. All porphyrins have four absorption 

 bands in the visible region between 500-700 mjU. The heights of these peaks relative to 

 each other vary with the particular structure. Stronger than any of these bands is the 

 so-called Soret band in the near ultra violet at about 400-450 m/i. Application of a low 

 dispersion, direct vision spectroscope to intact plant tissues has yielded many important 

 results and is still the most useful method for studying plant cytochromes. Details of 

 the procedures are well described by Hartree (19). Because of the low concentration of 

 hematin compounds in most plant tissues, rather thick sections must be used to observe 

 light absorption; a powerful light source is required; and the tissues may be infiltrated 

 with glycerol or pyridine to increase transparency. Since the bands of reduced cyto- 

 chromes are most distinct and informative, oxygen must be excluded or a reducing agent 

 such as sodium dithionite (hydrosulfite) added. Under these conditions a spectrum like 

 that of Figure 13-1 may be observed in, for example, an onion bulb: 



