\ H 7_ '7’ .7 _7’7' 7 -’77 i _#7 7_ 7 7 77 7 - 7- 7 _ 7 77 _ i 7_ 77 -7 i 7 f 7-7 7 7 77 A A A \ .401 IN—x _CDCD DEVELOPMENT OF A SYSTEM FOR THE STUDY OF THE BIOLOGICAL DEGRADATION OF CHLOROPHYLL Thesis for the Degree of M. S . MICHIGAN STATE UNIVERSITY ROGER F. McREETERS 1967 ; 31:72:13 LIBRARY Michigan State 5 University a: amome av 17 - unnsasons h I. max mom "1:74 ' LIBRARY muons “A. -Q-.-‘“. Ill ‘ W A H .t I‘VRRRRRRAHRR I 3 1293 00831 9059 JUN 0 3 2005 ABSTRACT DEVELOPMENT OF A SYSTEM FOR THE STUDY OF THE BIOLOGICAL DEGRADATION OF CHLOROPHYLL by Roger F. McFeeters ChlorOphyll a_was purified, suspended in water containing triton X-100, and injected into ripening green bell peppers (Capsicum frutescens). A 40-50% loss of the injected pigment was observed at the completion of ripening. The pigment loss was less than 5% when peppers were injected and extracted immediately. A maximum of 7% loss of chlorOphyll a was observed when the pigment was suspended in pH 5.4 buffer in the dark for periods up to two weeks. The amount of pigment loss was de- pendent upon the variety and the stage of ripening of the pepper. Enzyme extracts of peppers showed no ac- tivity in a system which bleaches chlorOphyll rapidly when legume seed extracts are used as a source of enzyme. These data indicate that the observed loss of injected chlorOphyll a is a result of a physiologically important chlorophyll degradation system. Roger F. McFeeters Labeled chlorophyll a, with a specific activity of 7-8x105 dpm/mg, was prepared from young wheat plants fed 14C02. The labeled chlorophyll a (0.2-0.3 mg) was injected into ripening peppers and the distribution of activity in pepper extracts after pigment degradation was observed. Significant activity was demonstrated in the acetone-water extract, remaining after transfer of lipid material to petroleum ether, within two days of injection, but the activity was nearly constant for the next 12 days of the experiment. The amount of radio- activity in the residue, obtained after acetone ex- traction of the pepper, and an 80% ethanol extract of this residue increased in both fractions throughout the experiment. The residue, which contained polysaccharide and ninhydrin positive material, had the largest amount of radioactivity of these three fractions at the con- clusion of the experiment. The degradation of labeled chlorOphyll §_by ripening peppers provides a tool for further studies of a pathway for the biological degradation of chlorOphyll. DEVELOPMENT OF A SYSTEM FOR THE STUDY OF THE BIOLOGICAL DEGRADATION OF CHLOROPHYLL BY Roger F. McFeeters A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Master of Science Department of Food Science 1967 ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. Sigmund H. Schanderl for his encouragement and guidance during my graduate and undergraduate studies. Thanks are due to Dr. Richard Ziegler for suggestions during the course of this research. ii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW Photodegradation. . . . . . . . . . . . . . . 4 ChlorOphyll degradation and lipid oxidation . 6 Enzymatic chlorOphyll degradation as a normal physiological process. . . . . . . . . 7 Possible enzymes and intermediates in chloro- phyll degradation . . . . . . . . . . . . . . 9 ChlorOphyllase. . . . . . . . . . . . . . 9 Pheophytin. . . . . . . . . . . . . . . . lO ChlorOphyllide. . . . . . . . . . . . . . ll PheOphorbide. . . . . . . . . . . . . . . 12 Other chlorOphyll-type compounds. . . . . 12 Degradation of chlorOphyll to smaller prOduCts O O C O O O O O O O O O O O O O 0 l4 METHODS AND MATERIALS Materials . . . . . . . . . . . . . . . . . . 18 Methods PheOphytin preparation. . . . . . . . . . l8 ChlorOphyll a preparation . . . . . . . . 19 Preparation of l4C-chlorophyll a. . . . . 21 Extraction of peppers . . . . . . . . . . 23 iii Determination of chlorophyll pheOphytin a . . . . . . . . 0"” m :3 Q; Thin-layer chromatography. Radioactivity measurements . . . . . . . . Injection of peppers . . . . . . . . . . . Bleaching of chlor0phyll 3 using Holden's system . . . . . . . . . . . . . . . . . . Time course of chlorophyll degradation in variety 035 peppers. . . . . . . . . . . . RESULTS AND DISCUSSION 0 O O O O O O O O O 0 O O 0 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . BIBLIOGRAPHY O O O O O O O O O O O O O I O O O O 0 iv 24 25 26 26 28 29 31 44 46 Figure LIST OF FIGURES Page Recovery of chlorOphyll a suspended in pH 5.4 acetate buffer. . . . . . . . . . 36 Recovery of chlorOphyll a from ripening 035 peppers as determined by spectro- photometric measurement. . . . . . . . . 39 The incorporation of radioactivity into various pepper fractions during degra- dation of injected chlorophyll a . . . . 40 Table LIST OF TABLES Page Degradation of chlorophyll a and pheophytin a in ripening 035 peppers left on the plant. . . . . . . . . . . . 32 Degradation of chlorophyll a by two varieties of peppers during ripening in darkness. . . . . . . . . . . . . . . 34 ChlorOphyll a recovery using pepper extract in Halden's chlorOphyll bleaching system . . . . . . . . . . . . 37 vi INTRODUCTION The breakdown of chlorOphyll is an obvious and widespread occurrence in nature as evidenced by the yellowing of leaves or the ripening of fruits. Therefore, an understanding of the causes of this degra- dation and the pathways it may take is of considerable intrinsic interest. Also, knowledge of the mechanism of chlorOphyll destruction would permit further investi- gations concerning its biological significance. The question of the existence of chlorophyll turnover in mature tissues has not been adequately answered (Perkins & Roberts, 1963) (Wickliff & Aronoff, 1963). A better comprehension of the process of chlorophyll breakdown would certainly aid experiments in this area. It would be possible to decide whether chlorophyll breakdown during senescence has some importance for the organism, such as formation of breakdown products with some physi- ological significance (Spencer, 1965), or whether the degradation is simply a side effect of other reactions and of little consequence to the organism. l Since the structure of chlorOphyll is very similar to heme, knowledge obtained about chlorOphyll metabolism is likely to find application in the study of heme metabolism. Control of color quality of foods is an important consideration in many areas of the food industry. Canned green vegetables have a less desirable color than fresh vegetables. Freezing these products preserves the color for a longer period of time, but color deterioration still occurs before other quality factors such as flavor or texture become unacceptable.‘ Sometimes bananas do not degreen, rendering them unsalable, which results in considerable losses. On the other hand, the shelf life of green peppers might be extended if degreening could be prevented.' Better solutions to these and similar problems would result from an understanding of the mecha- nism of chlorophyll degradation. An aspect of chlorOphyll degradation of which the food industry should be aware of is the toxicity of certain breakdown products. Tsutsumi and Hashimoto (1964) have shown that perpheophorbide was reSponsible for an occurrence of photosensitization in Japanese fishermen who had eaten abalone which had accumulated this compound in the liver tract. Patton & Benson (1966) have shown that in the bovine rumen phytol, the alcohol produced by deesterification of chlorOphyll by chlorophyllase in .yiggg, is converted to phytanic acid. Phytanic acid is known to accumulate in humans afflicted with Refsum's syndrome. These aspects of the problem of chlorophyll degradation illustrate the importance of learning the pathway of the breakdown. However, there is presently very little information available to aid understanding of the problem. Therefore, the efforts of this labo- ratory are being directed toward the elucidation of the mechanism and products of the biological degradation of chlorophyll. Lfi: L5 ‘“_‘ LITERATURE REVIEW Co (1966) has recently presented a good general review of chlorOphyll research. Therefore, this review will be confined to papers directly concerned with the t0pic of chlorOphyll degradation. Though the degradation of chlorOphyll is of con- siderable scientific and commercial interest, information on the topic is meager. At the present time there appear to be three types of chlorOphyll degradation which may be of biological significance. Photodegradation Sironval and Kandler (1958) exposed cells of Chlorella pyrenoidosa to very high light intensities and observed photobleaching of the pigments. At 100,000 lux there were two stages in the bleaching process. During a 30 minute induction period there was little chlorOphyll loss. This was followed by a period of rapid chlorOphyll bleaching. Oxygen is required for both stages of bleach- ing. The rate of bleaching of pigments present in the Chlorella was carotene > chlorophyll a > chlorOphyll b > carotenols. In a study of the metabolic changes which occurred during the induction period (Kandler and Sironval, 1959), it was concluded that the effects of light are similar to the effects of x-rays or ultra- violet radiation. The prOposal was made that the organism has a protective system which is destroyed during the induction period. The chlorophyll destruction itself is a secondary process which occurs after loss of the protective system. Below a light intensity of 70,000 lux the protective system apparently was able to c0pe with the stress since only small losses of chlorophyll were observed with prolonged light exposure. No attempt was made to elucidate the nature of the protective system, or to isolate and identify products of chloro- phyll destruction. Earlier Aronoff and Mackinney (1943) studied the photooxidation of chloroPhyll in acetone and benzene solutions. PheOphytins were formed either in the presence or absence of oxygen. PheOphytin a formed faster than pheOphytin b. However, oxygen was required for degradation to proceed beyond the pheOphytin stage. A spectrum was reported for some pink degradation products, but no further characterization of the compound(s) was done. In addition to the work which is needed to identi- fy products of photodegradation and the mechanism of the reaction, an evaluation of the importance of photo- destruction as a mechanism of chlorOphyll degradation in nature is needed. Chlorophyll Degradation and Lipid Oxidation Sumner (1942) showed carotene was destroyed during the enzymatic peroxidation of unsaturated fatty acids. Blain (1953) deve10ped an assay for soybean lipoxidase activity by measuring the extent of carotene bleaching. An unsaturated fatty acid, such as linoleic acid, was necessary for the reaction. Temperature, enzyme concentration, substrate concentration, time course, and pH dependence were obtained for the reaction. Wagenknecht et. a1. (1952) observed the degra- dation of chlorOphyll in frozen peas. The degradation was attributed to the action of lipoxidase. Walker (1964) came to similar conclusions about the loss of chlor0phy11 in frozen French beans. Holden (1965) made some modi- fications of Blain's carotene bleaching system and used it to bleach chlorOphyll. She found that extracts from a variety of legume seeds and plants could be substituted for soybean extract though the extent of bleaching varied. A number of saturated and unsaturated long chain fatty acids would substitute for linoleic acid, but short chain acids could not. Chlorophyll degradation accompanying fat peroxidation appears to be of considerable importance in some food products. Holden's system should provide a suitable model system for further study of the products of this reaction. However, there have been no reports that this type of degradation is responsible for chloro- ' phyll loss in tissue which has not been damaged by treatments like freezing or extraction.‘ There has been \ no report of this system being active in non-legumes. The lack of specificity of this degradation is evidenced by the fact that carotene is also degraded. Since carotenoid synthesis often accompanies chlor0phyll degradation in senescent tissues, it indicates that a carotenoid degradative system in these tissues is inactive or only slightly active. ‘Therefore, it appears unlikely that a lipoxidase related degradative system can be responsible for chlorOphyll loss during a period of carotenoid synthesis. AS'a result of these considerations, the possibility that such a system is a general degra- dative mechanism in plants appears unlikely. "'Enzymatic Chjorgphyll-Degradation as """" ‘a Normal'Physiological Process There is considerable circumstantial evidence for an enzymatic chlorophyll degradation which is physi- ologically important. It is well known for many fruits that ripening processes continue and chlorOphyll loss occurs in darkness. Corn seedlings degreen when placed in darkness (Frank & Kenney, 1955). Chlorella protothecoides bleaches in light or darkness when placed on a lOW‘nitrogen"medium’(ShihiraeIshikawa and Hase, 1964). Wolken (1961) reports that Euglena gracilis var, ‘bacillaris bleaches rapidly when placed in darkness. However, his experiments do not make it clear whether this is a true or apparent degradation because of di- lution of pigment by the culture growing in darkness. Hoyt (1966) has added considerable strength to the idea of an enzymatic chlorOphyll degradation. He observed that pieces of ryegrass (Lolium perenne) lose over 90% of their initial chlorophyll after a six day incubation in hygrostats at 25°C. If the grass is ground in a mortar, only a 25% chlorOphyll loss was ob- served. "Boiling the tissue before incubation results in only a 10% pigment loss. Chlorophyll degradation stOps when the moisture content of the grass falls below 33%. Cut ryegrass held at 5°C for six days lost 10% of its initial chlorOphyll. When it was returned to 25° and incubated for a second six day period, chlorOphyll breakdown resumed. After the incubation, 80% of the initial chlorophyll had disappeared. If this experiment is repeated, except that the tissue is frozen at -15° for the first six days, only slight chlorOphyll loss is observed after six days at 25°C.‘ These observations are all consistent with the idea of an enzymatic chlorophyll degradation. Moreover, in contrast to chlorOphyll de- struction related to lipid oxidation, this degradation system is easily damaged as shown by the inhibition caused by grinding or freezing the tissue. Possible Enzymes and Intermediates in Chlorophyll Degradation It can be concluded from the evidence presented above that an enzymatic system for chlorOphyll degradation exists in plants. The evidence for the participation of chlorophyllase in this pathway and the possibility that certain chlorOphyll derivatives are intermediates in the biodegradation will now be examined. ChlorOphyllase Since chlorophyllase was discovered (Willstatter & Stoll, 1913), there has been a question whether the enzyme is active during synthesis or degradation of chlorOphyll. A phytylation step is necessary to convert chlorophyllide to chlorOphyll. Until recently, except for a report of phytylation by Willstatter which was of questionable physiological significance, only the removal of phytol from chlorOphyll had been observed in yitgg (Klein & Vishniac, 1961). This tended to support a role for chlorOphyllase in chlorOphyll degradation. Experiments by Shimizu and Tamaki (1963) have demonstrated phytylation of chlorOphyllide a to form 10 chlorOphyll §_£gflyitrg by purified chlorOphyllase prepa- rations. The same authors (1962) found that chloro- phyllase activity increased prior to chlorOphyll increase in tobacco plants, and the decrease of activity was parallel with chlorOphyll loss in older plants. Sudyina (1963) reported similar results for the chlorOphyllase activity of tomato, strawberry, raspberry, apricot and cherry fruits. Therefore, the most likely physiological function of chlorOphyllase now appears to be in the biosynthetic rather than the degradative pathway of chlorOphyll. PheOphytin The magnesium ion is removed from chlorophyll a very readily under slightly acid conditions. When chlorophyll is extracted from plant tissues, pheOphytin is nearly always found even when great care is taken to neutralize the extraction medium (Bacon & Holden, 1967). Therefore, the question which must be decided is whether the pheophytin is a physiological degradation product, or whether it is solely an extraction artifact which cannot be avoided. The amount of pheophytin observed is in general proportional to the care used in extraction and chromatographic procedures. Very small amounts of pheOphytin are obtained when prOper precautions are taken to prevent its formation. Therefore, pheOphytin is not considered to be a naturally occurring chlorophyll ll derivative. The possibility does exist that degradation may occur through pheophytin, but the pheOphytin present at any time is too small to differentiate between that formed on extraction and that which occurs naturally. Chlorophyllide ChlorOphyllide is believed to be an intermediate in chlor0phyll biosynthesis (Bogorad, 1966). It can also be formed by the action of chlorophyllase ig‘yiggg. Proof that it is a physiological degradation intermediate ‘would consist of showing (1) that chlorophyllide is present in tissue which is degrading chlorophyll, (2) that the chlorophyllide observed is not a product of the biosynthetic pathway, (3) that the chlorOphyllide is not formed by the activity of chlorOphyllase in tissue injured during the experiment or during the extraction period. These conditions have not been met, so there is no conclusive evidence that chlorophyllide is a degra- dation product of chlorOphyll. The work of Shimizu and Tamaki (1962, 1963) and of Sudyina (1963) indicates that chlorophyllase is active in the biosynthesis of chlorOphyll. As a result, the probability that chlorophyllide is a physiological degra- dation intermediate is small. l2 Pheophorbide PheOphorbide is derived from chlorOphyll by :removal of both the magnesium ion and the phytyl group. Since removal of both the phytyl group and magnesium ion _;§'yizg is doubtful, it must be concluded that jpheophorbide is an unlikely intermediate in the bio- ciegradation of chlorophyll. Other Chlorophyll—Type Compounds Strain (1954) reported a number of conditions xvhich cause alteration of chlorophylls. The chlorophyll alteration products were called chlorophyll a', chloro- phyll _b_' ‘and allomerized chlorophyll. Recently a number of reports have appeared of (other products which are slightly different from chloro- jphyll in their chromatographic and spectral character— istics. ‘Michel-Wolwertz and Sironval (1965) described some products extracted from Chlorella, according to the chromatographic and spectral characteristics. They called these products chlorOphylls a and 2! a3! a4! a5! b4. Schanderl and Lynn (1966) reported spectra of several compounds obtained from thin—layer chromatograms of extracts of Capsicum frutescens at various ripening stages. ’Bacon (1966) maintained that many, if not all, of these pigments were artifacts of the method of chromatography. Bacon and Holden (1967) describe some pigments called "changed chlorophylls a-l, a-2, b-l, and l3 b-2" which are formed when leaves of barley (Hordeum vulgare EX' Maris Badger) and hogweed (Heracleum sphondylium L.) are subjected to various physical and chemical treatments. ~The authors do not think these pigments are present in normal plant tissue. Co and Schanderl (1967) reported the occurrence of chlorOphyll- type compounds in extracts of green bell peppers, banana peel and cucumber peel with blue absorption maxima at 418 and 444 nm. It was concluded that these compounds are not artifacts of chromatography. However, the possibility that these substances are formed even during very careful and rapid extraction procedures cannot be discounted. There have been no structures reported for any of these compounds. 'Since different extraction and chromatographic procedures were used in nearly every case and no correlations of the various methods have been made;‘it is possible that some of these alteration products are identical.‘ The occurrence of any of these compounds ig'yizg is viewed with considerable skepticism because of the possibilities of changes during ex- traction and'chromatography.‘ Even if some of these compounds occur in plant tissue, their relationship to chlorophyll degradation would need to be established. However, the fact that these products may be formed in damaged tissue or during chromatography does not prove they do not exist i£_vivo. H ’1“ . v‘M‘W-nfl“ l4 Degradation of ChlorOphyll to Smaller Products In the course of the degradative process it is expected that the porphyrin ring will be broken, and after a series of reactions, yield some compound(s) which is a common cellular metabolite. There are some difficulties to be met'when studying this problem which deserve mention. Any compound formed during the degradation will be present in small amounts. Even in very dark green higher plants like spinach,chlorophy11 content does not exceed 500 - 750 mg per kilogram fresh weight. If several compounds are formed, the amount of any particu- lar product may be only a few milligrams in a kilogram of tissue. This is no particular problem in the case of chlorophyll or its derivatives with an intact porphyrin ring because they have characteristic visible spectra which are well known.' In addition,unst of these compounds display strong pink or red fluorescence under ultraviolet ! E! A "light. This prOperty provides an extremely sensitive I method for detection of these compounds in solutions or ’on chromatograms. However, when the porphyrin ring is destroyed,these two properties are lost. The investi- ‘gator is left in the position of having to isolate unknown compounds,which are present in small amounts,with no method to detect and distinguish them as chlorophyll 15 degradation products unless labeled chlorOphyll could be used. Though no compounds have been found in biological systems which are known to be derived from chlorOphyll after cleavage of the porphyrin ring, it is possible to speculate on the general type of products which may result. A situation analogous to heme degradation may result in which there is a significant accumulation of linear pyrroles containing from one to four pyrrole units. These would differ in pr0perties from the pyrroles obtained from heme, but extension and modifi— cation of the methods used to detect heme degradation products might be expected to detect such compounds if they exist. Unfortunately the only linear pyrroles reported to occur in plants are'phycocyanins, phycobilins, and phytochrome which apparently are not connected with chlorophyll metabolism. 'Seybold (1943) found no compounds {an} obviously derived from chlorophyll in autumn leaves. Egle (1944) suggested Seybold's failure to find such products may have been due to the extreme speed of the reactions which convert chlorophyll to small compounds. If this is true, it might be expected that amino acids or organic acids appear as chlorOphyll degradation products. The isolation and identification of the degradation product in this situation would be relatively easy except that it would be difficult to 16 show whether any particular compound was derived from chlorophyll and not from some other reaction in the cell. In View of the experimental difficulties of the problem, a system was needed which would degrade exoge- nous chlorOphyll. Such a system would make it possible either to obtain a relatively large accumulation of 14C labeled chlorophyll degradation products, or to add and look for labeled products. Earlier experiments in this laboratory had shown no degradation, other than pheophytin or pheophorbide formation, when chlorophyll was added to isolated chloroplasts or to acetone powders prepared from ripening bananas or peppers. Chlorophyll degradation stopped even when pieces of ripening pepper were vacuum infiltrated with water. The conclusion was reached, and recently confirmed by Hoyt (1966), that the enzymatic degradation of chlorophyll was very sensitive to tissue damage and that the preparative procedures destroyed the degradation system. 'Mature green peppers lose their chlorOphyll very rapidly once the ripening process begins. No chlorophyll l or red fluorescent chlorophyll derivatives can be detected at advanced ripening stages (Schanderl and Lynn, 1966). Therefore, it was thought that the pepper might degrade chlorophyll or its derivatives if they were introduced into the tissue during the ripening period. An attempt 17 to develop a useful system for the study of the biologi- cal degradation of chlorophyll is described in this thesis. "K.T~ "1‘. A.Iflb’bl‘. All,“ ._.. -_- 2v-7 II. METHODS AND MATERIALS Materials A. Fresh spinach from a local food market was used as a source of chlorOphyll and chlorophyll derivatives. B. Two varieties of Capsicum frutescens, a dark green variety designated 044 and a light green variety designated 035, were grown in a green- house for use in these experiments. Methods A. PheOphytin preparation — Spinach leaves were ground in a Waring blendor with enough acetone to give a final concentration of 80% acetone. Oxalic acid was added after grinding. The solution was allowed to stand at room tempera- ture until the color changed from green to gray indicating that most of the chlorophyll had been converted to pheOphytin. The solution was filtered with a Bfichner funnel and the residue washed with acetone to remove most of the 18 19 remaining pigments. This solution was over- layered with ethen.and water or concentrated sodium chloride solution was added until the pigments were transferred to the ether layer which was then washed several times with water to remove acetone and water from the ether. The ether solution was dried in a flash evapo- rator to remove any remaining water. The pigments were dissolved in petroleum ether and applied to a column packed with powdered sugar containing 3% starch (Domino 10X). When the pigment mixture was absorbed to the t0p of the column, the column was developed using 3% acetone in petroleum ether. Pheophytin a was a gray band which moved behind a and B carotene on the column. After the pheOphytin band had moved almost to the bottom, the column was sucked dry. The pheophytin band was scraped out and the pigment eluted from the sugar with ether. This pigment was used for further experiments. ChlorOphyll a preparation — Fresh spinach leaves were used as a source of chlorOphyll. The ex- traction and separation method was essentially as described by Strain and Svec (1966). The spinach was placed in boiling water for 2 20 minutes. The water was then squeezed out of the tissue as completely as possible. The pigments were extracted with a 4:1(v/v) mixture of methanol-petroleum ether. This step was carried out three times to give nearly complete extraction. The pigments were transferred to petroleum ether by adding water to the methanol- petroleum ether extract. The petroleum ether layer was washed with water several times to remove residual methanol. Sometimes it was necessary to add some concentrated sodium chloride solution to prevent formation of emulsions. A small amount of diethyl ether was added to the petroleum ether solution to prevent precipitation of the pigments. This was applied to a 3 by 12 inch column packed with powdered sugar and the column was developed with 0.5% n-propanol in petroleum ether. The isolation procedure varied somewhat from Strain's procedure. Development of the column was stOpped when chlorophyll a had moved near the bottom. Chlorophyll a was scraped out, eluted from the sugar with diethyl ether and dried with a vacuum evaporator. It was re- dissolved in petroleum ether and washed with 50, 21 60, 70, 80, and 90% methanol to remove colorless compounds eluted from the sugar. A small amount of diethyl ether was added to the petroleum ether solution. This was applied to another 3 by 12 inch sugar column. The column for the second chromatography was developed with 5% acetone in petroleum ether. After development the chlorophyll was eluted as before. Use of a second solvent system was pre- ferable to Strain's procedure of rechromatography with the same solvent. The 0.5% n-propanol- petroleum ether solvent gives a better separation from carotenoids and chlorOphyll 2 than a 5% acetone-petroleum ether solvent, but the chlorophyll g_was often contaminated with a minor carotenoid pigment and with some pheophytin a which moved only slightly faster ram“ than chlorophyll a. Chromatography with the I acetone containing solvent gave a ready separation from these contaminants on the II a! second chromatography. A Preparation of l4C-ChlorOphyll a Two hundred wheat seeds were planted in sterile soil in an aluminum pie plate. The wheat was grown in dim light for 3-4 days until the primary leaves were 22 approximately 1 1/2" through the coleoptile tip. The wheat was placed into a desiccator and a 6 lb/per sq. in. vacuum was pulled. A small round bottom flask which contained three millicuries (11.5 mg) of Bal4CO3 had previously been connected to the desiccator. Four ml of 5N lactic acid was slowly added to the barium carbonate and the system was sealed completely until the 14CO2 generation was completed.' The flask was flushed with air until only a slight vacuum remained in the system. The desiccator was sealed and kept in a hood under constant light produced by two 30 watt fluorescent lamps at a distance of three feet until the wheat grew to 7-9 inches. This took 3-4 days. The wheat was cut and the chlorOphyll extracted according to the procedure described for chlorophyll a preparation. The only modifications made were that the wheat was ground slowly in a Sorvall omnimixer during the extraction and four or five ex- f'“ tractions with methanol-petroleum ether 4:1(v/v) were i made. The wheat tissue was more difficult to extract than spinach leaves so these changes were necessary to ; get a complete extraction. L7 ‘If the specific activity of the chlorophyll a remained constant after rechromatography and the chloro- phyll was spectrally pure, it was used for further ex— periments. One preparation'of the size described gave approximately 5 mg chlorophyll a with a specific activity 23 of 7-8x105 dpm per milligram assuming a counting efficiency of 11%. Extraction of Peppers Peppers were cut into pieces, placed in an omnimixer cup and acetone was added to give at least an 80% acetone concentration calculating the tissue weight as water.' The pepper was ground with the omnimixer for one minute and the ground material was filtered with suction through a fine fritted funnel. The residue was returned to the mixing cup and 100 ml acetone was added. It was ground for one minute and-again filtered through the fritted funnel. The residue was washed with acetone until the filtrate was free of pigment and all filtrates were combined in a separatory funnel. Approximately 100-150 ml of petroleum ether was added followed by 50 ml water.‘ The funnel was shaken gently until the petroleum ether and acetoneewater layers separated. The acetone- water layer was removed and the petroleum ether layer was washed several times with water to remove remaining acetone.w The petroleum ether solution was evaporated to dryness in a vacuum evaporator.’ It was usually necessary to add acetone about three times to aid evaporation of a small amount of water which remained in the petroleum ether.‘”These dried pigments were made to a known volume with either diethyl ether or acetone for pigment determinations or other analysis. Experience has shown 24 that at least 95% recovery of the remaining pigment was normally obtained. Determination of ChlorOphyll a andIFheOphytin a All quantitative absorbance measurements were made using a Beckmann DU spectrOphotometer with a Gilford readout attachment. For determination of the concentration of stock solutions of chlorophyll a diethyl ether was used as the solvent. The absorptivity value of 100.9 1 g-lcm-1 at 662 nm (Smith & Benitez, 1955) was used to calculate the pigment concentration.“ The ratio of the absorbance at 429 nm to the absorbance at 662 nm was used as an indication of the spectral purity of the chlorOphyll a. Chlorophyll a in most experiments was exposed to slightly acid conditionS'which caused a partial con— version to pheOphytin a. To avoid the complication of measuring a mixture of two pigments, the chlorophyll a was completely converted to pheophytin a by adding 2 mg/ml oxalic acid to the solutions for three hours before readings were taken (Vernon, 1960). Absorbance readings were made at 667 nm, the red absorption maximum of pheOphytin, and at 700 nm to control the light scattering of the solution. The difference between these readings was used to calculate the pheOphytin concentration according to Beer's law. The absorptivity is 25 58.7 1 g-lcm-l (Wilson et. al., 1962). If pheOphytin a was injected into a pepper, the same procedure was followed except the oxalic acid was not added to the solution. The chlorOphyll a concentration was found by multiplying the pheOphytin a concentration by 1.025, the ratio of the molecular weight of chlorOphyll a to the molecular weight of pheophytin a. For some experiments 80% acetone was used as the solvent. In this case the same readings and calculations were made except that the absorptivity for pheophytin a -1 not corrected for magnesium loss is 55.2 1 g-lcm (Vernon, 1960). Thin-layer Chromatography Co and Schanderl (1967): Plates were coated with silica gel G (E. Merck, Darmstadt, Germany) using a Desaga spreader. Thirty grams of silica gel were mixed with 60 ml of water to make five 20 x 20 cm plates 0.25 mm thick. The plates were dried at room temperature. The solvent system was benzene-petroleum ether-acetone (10:2.5:2, v/v/V). L Hager and Bertenrath (1962): Five 20 x 20 cm 0.25 mm thick plates were made by mixing 12 g kieselgur G, 3 g kieselgel, 3 g CaCO 3 3, and 0.02 g Ca(OH)2 with 50 ml 8 x 10- M ascorbic acid solution and spreading with a Desaga spreader. The plates were dried for l l/2 hours at 50-60°C. A solvent system consisting of 100 ml 26 heptane, 12 ml iSOprOpanol and 0.25 ml water was used for development. Schneider (1965): Fifteen grams MN cellulose 300 (Macherey, Nagel & Co.) were mixed with 100 ml water in an omnimixer. Five plates (20 x 20 cm) 0.4 mm thick were made using a Desaga spreader. The development solvent was methanol-dichloromethane-water (100:18:20, v/v/v). Bacon (1965): Plates were made as with the Schneider method. The development solvent was petroleum ether- acetone-n-propanol (90:10:0.45, v/V/v). Radioactivity Measurements A Nuclear Chicago planchet counter was used for all radioactivity measurements. For some experiments it was adapted for low background counting. The counting procedure varied and is described for each experiment in . . . . lam Wthh radioactiv1ty measurements were made. FL ‘Injection‘of Peppers Several different methods of pigment injection were used. For the first experiments a colloidal sus- pension of pheophorbide a was prepared by slowly adding ‘drops of an acetone solution of the pigment to water while stirring on a magnetic stirrer. PheOphorbide a 'was used because it is more hydrOphilic than chlorophyll a'or pheOphytin a and, consequently, gave a better 27 suspension. The aqueous suspension was injected into the hollow of the peppers with a sterile syringe and dried on the endocarp. This water suspension method had the disadvantage that a quantitative injection of the pigment was very difficult because some of the pigment would adhere to the beaker or stirring bar when it was drOpped into the water. A quantitative injection of pigments was possible by injecting a 60-70% acetone solution of chlorophyll 3. Even though the high acetone concentration caused some injury to the pepper, degradation of the pigment was still observed. The best method found for the injection of chlorOphyll a was to dissolve the chlorophyll in 0.2 ml acetone in a test tube and suspend it in 3-5 ml of water containing 0.2% triton X-100. The chlorOphyll suspension was injected into the pepper with a sterile syringe. Test tube and syringe were washed with 0.1 m1 acetone and 0.5 ml water containing triton X-100, which were also injected into the pepper. With this method a quantitative injection was accomplished with no visible damage to the pepper tissue. 28 Bleaching of ChlorOphyll 3 Using HoldenTs_System The possible presence of a chlorophyll degra- dative system in peppers similar to that found by Holden (1965) in legume seeds was checked by the following ex- periment. A variety 035 pepper of intermediate ripeness (about 1/4 of the surface orange) was cut into small pieces after removing the stem and seeds. Forty grams of tissue was ground in‘a mortar with 120 m1 of acetate buffer pH 5.9. This extract was centrifuged for 10 minutes at 5000 x G. The supernatant was used as the enzyme solution. The substrate consisted of 0.35 mg chlorOphyll a dissolved in 0.3 ml acetone. Sufficient acetate buffer pH 5.9 containing 0.2% triton X-100 was added to suspend the chlorophyll and give a final sample volume of 25 ml. Either 1 ml of 0.5% linoleic acid in ethanol or 1 ml ethanol was added to the suspension. The reaction was startedby adding enzyme extract and vigorously stirring the reaction mixture for 2 minutes. It was stopped after 15 minutes by adding 25 ml acetone. The mixture was transferred to a separatory funnel and 25 m1 petroleum ether was added. The funnel was shaken until the chlorophyll a was transferred to the petroleum ether phase. The acetoneewater phase was discarded. After several washings with water, the petroleum ether was evaporated to dryness. The chlorOphyll was taken up 29 and made to 25 ml with 80% acetone. The chlorophyll a remaining was determined by the method described above. Time Course of Chloro h 11 Degradation in Variety 035 Peppers Twelve 035 peppers of equal ripeness were each l4C-chlorOphyll a suspended in 5 injected with 0.26 mg ml 0.2% triton X-lOO. A small portion of the surface was orange indicating the beginning of ripening at the time of injection. The peppers were incubated in the dark at 26-27°C. At six different time intervals two peppers each were extracted and the petroleum ether extract was made to 25 ml with acetone. One pepper showed irregular ripening and was discarded. Chlorophyll a was determined according to the procedure described. The per cent recovery of the injected chlorophyll a was calculated. The acetone-water phase, obtained after the pigments were transferred to petroleum ether, was evapo- rated and made to*a final volume of 10 ml with water. The residue, remaining after acetone extraction, was boiled with 150 m1 of 80% ethanol for 1 hour. The mixture was filtered hot through a fine fritted funnel. The filtrate was evaporated and made to 10 ml with water. The residue was dried and weighed. The total activity in each of these four fractions was measured using a Nuclear Chicago low background 30 planchet counter. For all samples the average counting rate of five determinations on a known amount of sample was calculated. For the petroleum ether extracts some of the counting rates obtained were greater than would be expected if all the activity were recovered in this fraction. This may have been a result of evaporative concentration of the acetone solutions during storage. The residue was a powdery material. Therefore, to prevent contamination of the counting chamber the planchets were covered with a thin plastic wrap. The average loss of counts caused by the plastic wrap was determined to be 44% by counting several samples of chlorOphyll a before and after covering the planchets. The counting rates obtained from the residue samples were adjusted to correct for this loss. RESULTS AND DISCUSSION In a preliminary experiment a colloidal sus- pension of pheophorbide a was injected into variety 035 peppers, which contain little chlorophyll and are yellow- green in color, at an early ripening stage. When ripen- ing was completed, the pepper was extracted and the extract chromatographed using the procedure of Co and Schanderl (1966). Pheophorbide was not detected on the chromatogram, therefore, it was concluded that the pepper tissue degraded the injected pigment. Confirmation of this observation required a quantitative measurement of the injected and recovered pigment. The procedures used to obtain a quantitative injection, extraction, and estimation of the pigments ‘ are described in the methods section. Peppers variety 035 at an early ripening stage were injected with chlorophyll a and pheophytin a and allowed to ripen on the plant in the greenhouse with normal periods of daylight. The pigment degradation observed is shown in Table l. 31 32 TABLE l.--Degradation of ChlorOphyll a and PheOphytin a in Ripening 035 Peppers Left on the Plant. Amount Amount Compound Injected Recovered Per Cent Injected mg mg Degradation PheOphytin a 0.23 0.075 67 PheOphytin a 0.23 0.082 64 Chlor0phyll a 0.42 0.13 69 Chlorophyll a 0.42 0.065 84 A large amount of the injected chlorOphyll was degraded. However, it was possible that all or most of the degradation was a result of photodegradation of the pigment. To test this possibility chlorophyll §_was suSpended in two test tubes with 0.2% triton X-100. A hole was cut in the shoulder of a ripening 035 pepper and a test tube containing suspended chlorophyll was inserted. Another test tube was kept in the dark for a comparable amount of time. When the pepper ripened the chlorOphyll suspension was extracted with petroleum ether. The extract was dried and made to volume with acetone. A spectrum of this solution showed no chloro- phyll a present. Determination of the chlorOphyll a kept in the dark after the same extraction procedure showed a 104% recovery of the suSpended chlorOphyll a. This experiment indicated that the observed degradation 33 may have been a result of photodegradation of the in- jected pigments rather than a degradation by the tissue enzymes. It was necessary to show degradation of injected pigment in the absence of light before this system would be useful to study the biological degradation of chloro- phyll. ChlorOphyll a was injected as before except the peppers were picked and placed in the dark until ex— tracted. In addition to the yellow-green 035 peppers, some very dark green variety 044 peppers were also in- jected. The degradation observed in these peppers is shown in Table 2. The data in Table 2 demonstrate the character- istics of the degradation. Variety 035 peppers when in- jected at an early ripening stage degraded part of the chlorophyll 3, though the degradation was not as large as that observed in the light. Therefore, though some photodegradation occurred in the previous experiment, the pepper tissue was still responsible for a significant amount of degradation. Variety 044 peppers degraded only a small portion of the injected chlorOphyll. Both varieties when injected at later stages of ripening caused very small chlorOphyll losses indicating that fully ripe peppers are no longer capable of carrying on chlorOphyll degradation. When a ripe pepper was injected with chlorOphyll a and immediately extracted, only a 2% 34 TABLE 2.--Degradation of Chlorophyll a by Two Varieties of Peppers During Ripening In Darkness Per Cent Degra- Pepper Stage of Ripening at In- dation of Injected Variety jection and Extraction* Chlorophyll a 035 l 46 035 l 32 044 l 15 044 l 4 035 3 10 035 2 6 044 2 6 044 4 2 *l. Injected at early ripening stage; extracted at completion of ripening. 2. Injected when ripe; extracted after 5 days. 3. Injected at 2/3 ripeness; extracted at completion of ripening. Injected when ripe; extracted immediately after injection. loss of chlorOphyll was observed. This shows that the losses observed in the 035 peppers injected at the beginning of ripening did not occur during the extraction procedure. The fact that the amount of degradation de- pended upon the pepper variety and that chlorOphyll a could be left inside a ripe pepper for 5 days with little degradation indicated that the degradation was 35 not a result of air oxidation of chlorOphyll dried on the endocarp of the pepper. The pH of the juice squeezed from a green 035 pepper is approximately 5.5. Therefore, it seemed a reasonable possibility that the observed breakdown was caused by some acid catalyzed reaction and did not de- pend upon enzymatic activity in the pepper. Chlorophyll a (0.26 mg) was suspended as before except that 5 ml acetate buffer at pH 5.4 was used in place of water. The suspended pigment was kept in the dark at 26-27°C in test tubes. At six sampling times contents of three test tubes were transferred to 25 ml volumetric flasks and made to volume with acetone to give a final acetone concentration of 80%. The chlorophyll remaining was determined as described. The average of three determi- nations is plotted in Figure 1, expressed as per cent chlorOphyll recovery at zero time recovery. The results show that a significant chlorOphyll loss is only observed after 11 days and the 7% loss observed is not nearly as large as the degradation observed in the peppers. Another reason for the chlorOphyll degradation observed in peppers could have been a reaction caused by enzymatic lipid oxidation (Holden, 1965). If this were true,it would indicate that the degradation was a result of tissue injury. In this case Holden's degradative 36 w m g \ —O 8 904 m _ m ml 3 N 80 m __ 8 a: O t—‘l 5 7o ._ E4 Z m 84’ m 60 m J L I J 0 2 5r 8 IE 14‘ TIME (days) Figure 1: Recovery of chlorOphyll a suspended in pH 5.4 acetate buffer.* * Recovery expressed as percent chlorophyll at zero time. 37 system would probably be a better model system with which to study the reaction.' This possibility was tested by substituting an extract of tissue from a ripening pepper for the soybean extract in her system. The de- tails of this experiment were described and the results of the experiment are shown in Table 3. The chlorOphyll recovery is expressed as per cent of the control which contained neither enzyme extract nor linoleic acid. TABLE 3. --Chlorophyll a Recovery Using Pepper Extract in Holden's ChlorOphyll Bleaching System ml of Enzyme Linoleic Chlorophyll 3 Extract Acid Recovery* 0 - 100 0 + 101 10 - 104 10 (boiled) + 101 10 + 100 5 + 97 *Average of two reactions. Variation between two samples was always less than 2%. The largest chlorophyll loss was only 3% for a 15 minute incubation and is considered to be within experimental error. ‘Holden observed a 69% chlorOphyll degradation during a 2 minute incubation when soybean 38 extract was used. If such a chlorOphyll bleaching system exists in pepper tissue, it is either much less active than the soybean enzyme or it is active under different conditions. Blain et. a1. (1953) showed that carotenoids are also bleached under these conditions. Since a large increase in carotenoids is observed during ripening of peppers (Curl, 1964), this is one argument against such a bleaching system being active in the tissue. "It was concluded that the observed degradation of the injected chlorophyll probably occurs by some pathway other than that active in Holden's bleaching system. Information about the products derived from the chlorOphyll degraded by the peppers could be obtained using labeled chlorophyll.“ Chlorophyll a, assumed to be randomly labeled with carbon-14 (Perkins & Roberts 1962), was prepared and injected into twelve 035 peppers. The chlor0phyll recovery, determined by spectrophoto- metric measurement, expressed as per cent recovery of injected chlorophyll is shown in Figure 2. Figure 3 gives the total counts per minute of the acetone-water, 80% ethanol, and residue fractions. ‘The counting procedures were subject to errors which did not allow a quantitative measurement of activity. The residues were powdery and the size of the particles varied with the ripeness of the pepper. PERCENT OF INJECTED CHLOROPHYLL a RECOVERED |_n O O 80 6O 40 20 39 0 C G .1 v 1 I 1 .1 o 2 5' TA I1' 14' TIME (days) Figure 2: Recovery of chlorophyll a from ripening 035 peppers as determined by spectrOphotometric measurement. IN FRACTION TOTAL COUNTS/MIN. Na— Figure 3: 40 J 5' 8‘ 1f 141 TIME (days) The incorporation of radioactivity into various pepper fractions during degradation of injected chlorOphyll a. Each point repre- sents the avErage of five samples. A— —A , residue; 0 o, acetone-water; + ----- +, 80% ethanol 41 Therefore, there was undoubtedly some loss from sample self-absorption. The same problem of self—absorption was also present for the acetone-water and 80% ethanol extracts. However, much less material was on the planchets for these extracts than for the residue, so the self-absorption was probably much less. Therefore, the curves in Figure 3 give an estimate of the relative incorporation of activity into the fractions with time, but the absolute incorporation is likely to be somewhat greater especially in the residue fractions. The distribution of radioactivity in the differ- ent extracts gives some'idea of the types of products which are formed from chlorophyll.’ The combined activity in the acetone-water, 80% ethanol, and residue fractions in general is considerably less than would be expected if all the products formed from the chlorOphyll a which disappeared in the spectrOphotometric determination had appeared in these fractions.' Therefore, either activity 14 was lost aS" CO 'or some lipid soluble product(s) was 2 formed which remained in the petroleum ether extract. The activity in the acetone-water extract increased rapidly after injection and remained relatively constant after the second day. This fraction contains components of the pepper which are soluble in 90% acetone, but are hydrophilic enough not to be transferred to the petroleum ether layer.' The 80% ethanol extract contains the 42 material from the pepper which is insoluble in 90% acetone, but soluble in boiling 80% ethanol. The residue after hot 80% ethanol extraction consists of polysaccha- ride and ninhydrin positive material. The activity in both of these fractions increases with time. The next step in the study of this system is to isolate and identify the products formed from chlorophyll 3. Some attempts have been made to isolate products from the petroleum ether and acetone-water extracts by thin-layer chromatography with the methods of Co and Schanderl (1967), Hager and Bertenrath (1962), Schneider (1965), and Bacon (1965). In each case the extract was streaked on the origin of the chromatogram. After development the whole area of the chromatogram was divided into several different bands. Each band was scraped off the plate and eluted with a suitable solvent such as acetone or methanol. Visible spectra were made on the eluate from each band and the radioactivity of each eluate was checked. For petroleum ether extracts, radioactivity was associated with the pheophytin band. A small amount of activity was generally found in other bands, however, no distinct concentrations of activity were observed. There are two explanations for the inability to find distinct radioactive bands. The methods used were not apprOpriate for separation of the chlorOphyll degra- dation products formed or the plates were too overloaded 43 to give a good separation. The amount of carotenoids in ripe pepper extracts is much greater than the amount of injected chlorophyll. Therefore, it was necessary to overload the chromatograms with carotenoids to permit the detection of minor radioactive components if they occurred. Attempts to isolate radioactive compounds from the acetone-water extract have also been generally unsuccessful." Copper pheOphorbide a was isolated and identified from one pepper extract. This is a green non-fluorescent compound formed by the insertion of copper into the center of the porphyrin ring of pheOphorbide'a (Schanderl, 1962). Subsequent attempts to isolate it from the acetoneéwater extracts of other peppers injected with labeled chlorOphyll a were unsuc- cessful. This compound is known to be formed during processing and storage of green vegetables when suf- ficient copper is available. It is possible that by chance the pepper or extract was contaminated with some copper in the case where copper pheophorbide was observed. There may also be sufficient copper normally present in the tissue to form the c0pper pheophorbide, but if this were the source of the copper, it is not known why for- mation did not occur in all cases. No work has been done to isolate products from the 80% ethanol extract or the residue fraction. SUMMARY AND CONCLUSIONS The study of the biological degradation of chlorOphyll beyond the destruction of the porphyrin ring is presently in a primitive state of development. The study of this problem is difficult because very small quantities of unknown compounds must be isolated, identi- fied, and their origin established. A degradative system, which overcomes some of these problems, has been de- veloped for use in future studies of this problem. Ripening bell peppers (Capsicum frutescens) variety 035 were found to degrade injected chlorOphyll a. The data presented were consistent with the theory that the observed degradation was accomplished by a physiologically important mechanism. This degradation is believed to be different from that observed by Holden (1965) with a system containing linoleic acid and soybean extract. Labeled chlorophyll a was prepared and injected into ripening peppers for different periods of time. The distribution of radioactivity in three fractions ex- tracted from the pepper was established. Preliminary 44 45 chromatography of two fractions did not permit isolation of radioactive products. This system permits a more direct study of the degradation of chlorOphyll than was previously possible. The identification of small quantities of unknown compounds is still a problem, but the use of labeled chlorOphyll makes isolation and proof of their origin from chlorophyll considerably easier. Proof that this is a physiological system will have to wait until some compound which is formed from the exogenous chlorophyll is demonstrated $2.2iX2' and its appearance is correlated with the loss of endogenous chlorOphyll. BIBLIOGRAPHY Aronoff, S. and Mackinney, G. 1943. The photooxidation of chlor0phyll. J. Am. Chem. Soc. g;, 956. Bacon, M. F. 1965. Separation of chlorOphylls a and b and related compounds by thin-layer chromato- graphy on cellulose. J. Chromatog. 11, 322. Bacon, M. F. 1966. Artifacts from chromatography of chlorophylls. Biochem. J. 101, 34c. Bacon, M. F. and Holden, M. 1967.' Changes in chloro- phylls resulting from various chemical and physical treatments of leaves and leaf extracts. Phytochem. g, 193. Blain, J. A., Hawthorn, J., and Todd, J. P. 1953. The bleaching of carotene by‘a lipoxidase linoleate system. J. Sci. Food Agr. 4, 581. Bogorad, L. 1966. The biosynthesis of chlorophylls. In "The Chlorophylls", Vernon, L. P. and Seely, Go R0, edSo' pp. 481-510, Academic Press, N. Y. Co, D. Y. C. Lynn.‘ 1966. Detection, isolation, and characterization of chlorOphylls and related pigments during ripening of fruits and vegetables. Doctoral thesis, Michigan State Univ. Co, D. Y. C. Lynn and Schanderl, S. H. 1967a. Sepa- ration of chlorOphylls and related plant pigments by‘twoedimensional thin-layer chromatography. J. Chromatog. 26, 442. C0, D. Y. C. Lynn and Schanderl, S. H. 1967b. The occurrence of 418 and 444 nm chlorophyll-type compounds in some green plant tissues. Phytochem. g, 145. 46 47 Curl, A. L. 1964. The carotenoids of green bell peppers. Agr. Food Chem. 12, 522. Egle, K. 1944. Untersuchungen fiber die Resistenz der Plastidenfarbstoffe. Bot. Archiv. 42, 93. Frank, S. and Kenney, A. L. 1955. Chlorophyll and carotenoid destruction in the absence of light in seedlings of Zea mays L. Plant Physiol. 30, 413. I —" ' "" Hager, A. and Bertenrath, T. 1962. Verteilungschromato- graphische Trennung von Chlorophyllen und Carotinoiden grfiner Pflanzen an Dfinnschichten. Planta 58, 564. Holden, M. 1965. ChlorOphyll bleaching by legume seeds. J. Sci. Food Agr. 16, 312. Hoyt, P. B. 1966. Chlorophyll-type compounds in soil. I. Their origin. Plant and Soil XXV, 167. Kandler, O. and Sironval, C.‘ 1959. Photooxidation processes in normal green Chlorella cells. II. Effects on metabolism. 'Biochim. Biophys. Acta 33, 207. Klein, A. O. and Vishniac W.‘ 1961. Activity and partial purification of chlorOphyllase in aqueous systems. J. Biol. Chem. 236, 2544. Michel-Wolwertz, M. R. and Sironval, C. 1965. On the chlorophylls separated by paper chromatography from Chlorella extracts. Biochim. Biophys. Acta 94, 330. Patton, S. and Benson, A. A.‘ 1966. Phytol metabolism in the bovine. 'Biochim. BiOphys. Acta 125, 22. Perkins, H. J. and Roberts, D. W. A. 1963. On ‘chlorophyll turnover in monocotyledons and dicotyledons. Can. J. Bot. 41, 221. Schanderl, S. H. 1962. Metal complexes of chlorOphyll in vegetable tissues and model systems. Doctoral "thesis, Univ. of California, Davis. Schanderl, S. H. and Lynn, D. Y. C. 1966. Changes in chlorOphylls and spectrally related pigments during ripening of‘Capsicum'frutescens. J. Food Sci. 31, 141. 48 Schneider, H. A. W.’ 1966. Eine einfache Methode zur Dfinnschicht chromatographischen Trennung von Plastidenpigmenten. J. Chromatog. 21, 448. Seybold, A. 1943. Autumn leaf coloring. Botan. Arch. 44, 551. Shihira-Ishikawa, I. and Hase, E. 1964. Nutritional control of cell pigmentation in Chlorella protothecoides with Special reference to the degeneration of chlorOplast induced by glucose. Plant and Cell Physiol. 5, 227. Shimizu, S. and Tamaki, E. 1962. ChlorOphyllase of tobacco plants I. Preparation and properties of water soluble enzyme. 'Botan. Mag. (Tokyo) 12, 462. Shimizu, S. and Tamaki, E. 1963. Chlorophyllase of tobacco plants II. ‘Enzymic phytylation of chlorophyllide and pheophorbide in vitro. Arch. Biochem. BiOphys. 102, 152. Sironval, C. and Kandler, O. 1958. Photooxidation processes in normal green Chlorella cells. I. The bleaching process. 'Biochim. Biophys. Acta 29, 359. Smith, J. H. C. and Benitez, A. 1955. ChlorOphylls: Analysis in plant materials. In "Moderne Methoden der Pflanzenanalyse", Paech, K. and Tracey, M. V., eds., IV, pp. 142-196, Springer, Berlin. Spencer M. 1965. Fruit ripening. In "Plant Bio- chemistry", Bonner, J. and Varner, J. E., eds., pp. 793-825, Academic Press, N. Y. Strain, H. H. 1954. Oxidation and isomerization reactions of the chlorophylls in killed leaves. Agr. Food Chem. 2, 1222. Strain, H. H. and Svec, W. A. 1966. Extraction, separation, estimation, and isolation of the chlorOphylls. In "The Chlorophylls", Vernon, L. P. and Seeley, G. R., eds., pp. 21-66, Academic Press, N. Y. Sudyina, E. G. 1963. ChlorOphyllase reaction in the last stage of biosynthesis of chlorOphyll. Photochem. and Photobiol. 2, 181. 49 Sumner, R. J.’ 1942.’ Lipid oxidase studies. III. The 'relation between carotene oxidation and the enzymatic peroxidation of unsaturated fats. J. Biol. Chem. 21g, 215. Tsutsumi, J. and Hashimoto, Y. 1964. Isolation of perpheophorbi e‘a as a photodynamic pigment from the liver‘ofIabalone, Haliotis discus hannai. Agr. Biol. Chem. 23, 467. Vernon, L. P. 1960.‘ Spectrophotometric determination 'of chlorophylls and pheOphytins in plant ex- tracts. Anal. Chem. 22, 1144. Wagenknecht, A. C., Lee, F. A. and Boyle, F. P. 1952. The loss of chlorophyll in green peas during frozen storage and analysis.' Food Res. 21, 343. Walker, G. C. 1964. Color deterioration in frozen French beanS‘(Phaseolus vulgaris). J. Food Sci. 22, 383. Wickliff, J. L. and Aronoff, S. 1963. Turnover of chlorophyll a in mature soybean leaves. In "Studies on Microalgae and Photosynthetic Bacteria," pp. 441-449, Univ. of Tokyo Press, Tokyo, Japan. Willstatter, R. and Stoll, A. 1913. "Untersuchungen fiber Chlorophyll", Springer, Berlin. "Investigations on Chlorophyll", translated by Schertz, F. M. and Merz, A. R. 1928. The Science Press Printing Company, Lancaster, Pa. Wilson, J. R., Nutting, M-D. and Bailey, G. F. 1962. Use of tetracyanoethylene for removal of visual carotenoid spectra from solutions of pheOphytins. ‘Anal. Chem. 22, 1331. Wolken, J. J. 1961. "Euglena", Rutgers Univ. Press, New Brunswick, N. J. HICHIGQN STATE UNIV. LIBRQRIES ‘ III II III! 1 312930083 9059