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Porphyrins in the perienteric fluid of Ascaris lumbricoides
PORPHYRINS IN THE PERIENTERIC ‘4~C~~~~ ~~~~R~C~~~E~ G. D. Department
CAIN
FLUID
OF
and FERN BASSOW
of Zoology, The University of Iowa, Iowa City, Iowa 52242, U.S.A. (Received 23 April 1975)
Abstract-CAIN
G. D. and BASSOW F. 1976. Porphyrins in the perienteric fluid of Ascarfs ~~~~~~~c~~~e~. t~rern~~io~zul Journal fur Parusitafogy 6: 79-82. Porphyrins in the perienteric fluid of adult female A. lumhricoides were esterified in methanolic H,SOI, extracted in chloroform, separated by thin-layer chromatography, and identified spectrophotometrically before and after conversion to their zinc and copper chelates. Protoporphyrin IX was the major component, comprising 95.4% of the total; the remaining 4.6 ‘Awas coproporphyrin III. Uroporphyrin was not detected; no porphyrins were recovered from other worm tissues. Fluid from worms with light and dark colored guts varied in protoporphyrin content from 0.58 to 4.08 nmoles/ml, respectively, but fluid from both groups contained similar molar ratios of protoporphyrin, coproporphyr~n and heme. INDEX KEY WORDS: Ascaris lumbricoides; perienteric fluid; porphyrin; protoporphyrin IX; coproporphyrin III; thin-layer chromatography.
effect
in cultured
methyl ester;
that parasitic helminths lack the ability to synthesize porphyrin (Smith & Lee, 1963; Fairbairn, 1970; von Brand, 1973). It is therefore surprising that coproporphyrinogen oxidase, the penultimate enzyme in the porphyrin synthesis pathway (Sano & Granick, 1961; Tait, 1968) is evidently present at low activity levels in A. fumbr~coi~es mitochondria (Cain, in press). This finding suggested that the porphyrin pathway might be at least partially operative and prompted a search for the biosynthetic intermediates, uro-, copro- and protoporphyrin in A. lumbricoides.
IRON-PORPHYRIN complexes are common constituents of parasitic helminths, where they are found both in hemoglobins (for review, see Lee & Smith, 1965) and cytochromes (Cheah & Chance, 1970; Cheah, 1968; Hill, Perkowski & Mathewson, 1972; Cheah & Pritchard, 1975). Despite the wide occurrence of heme proteins in helminths, however, very little is known about the origin and metabolism of heme and porphyrin in these organisms. Nutritional studies with nematodes suggest that heme and porphyrins have a growth-promoting effect in Nippostr~ngy~us brasiliensis (Bolla, Weinstein & Lou, 1972, 1974), and in a free-living species, ~~en~rt~abdit~~briggsae (Hieb, Stokstad & Rothstein, 1970). The nature of the effect is poorly understood, but in at least one species, Ascaris lumbricoides, administration of hemes and certain other porphyrins and metalloporphyrins under in viva (Cain & Welshman, 1973) and in vitro (Smith & Lee, 1963) conditions causes an increase in hemoglobin production. In parasitic flatworms, heme has a growth stimulating
porphyrin
MATERIALS
AND METHODS
Adult female A. ~~mbric~jdeswere obtained at a local slaughterhouse and immediately transported to the laboratory. Worms were washed thoroughly in Harpur’s saline solution (Harpur, 1962) and dissected carefully so as not to contaminate tissues with worm gut contents. Muscle and reproductive tissues were washed in three changes of saline; guts were washed free of contents by flushing the lumens exhaustively with saline by means of a Pasteur pipette. Porphyrins in lyophilized tissues and perienteric Ruid were either extracted as free porphyrins using the method of Dresel & Falk (1956) or esterified with methanolic H,SO$ and extracted according to the methods of Doss (1971). Free porphyrins were separated by paper chromatography (Falk, 1961) and porphyrin methyl esters by thin-layer chromatography (TLC) on silica gel H (Doss, 1967). The TLC plates were scanned on a Turner model 111 fluorometer as described by Doss, Ulshofer & PhillipDormston (1971) and then photographed (Ulshofer & Doss, 1969). Quantification of porphyrin esters by fluorometric scanning (Doss et al., 1971) was not per-
Hymertolepis microstoma
(Seidel, 19711, even though this tapeworm apparently does not contain hemoglobin. Nutritional studies on trematodes are lacking, but the heme precursor, Saminolevulinic acid, was not incorporated into hemoglobins of Fascioiopsis buski and Philophthalmus megalurus under conditions where Iabelled amino acids were heavily incorporated into the protein moiety (Cain, 1969). The above results are usually interpreted to mean 79
80
G. D. CAIN and FERN BASSOW
formed, since fatty acid methyl esters, abundant in worm samples, tended to cochromatograph with protoporphyrin dimethyl ester and interfered with its fluorescence. Fluorescent porphyrin esters corresponding in chromatographic mobility to authentic standards (Sigma Chemical Co., St. Louis, MO.) were scraped from TLC plates and eluted from the silica gel with chloroform, while free porphyrin spots were eluted with 1 N-HCI following paper chromatography. Eluted substances were scanned in a Beckman Acta III spectrophotometer and quantified as free porphyrins and methyl esters (Falk, 1964) and as zinc and copper chelates of the methyl esters (Doss, 1971). Heme dimethyl ester, which remained at the origin under the TLC conditions em-
I.J.P. VOL.6. 1976
URO III
COPRO III
PROTOIX
ployed, was eluted from the gel with ethyl acetate and quantified, after appropriate dilution, by measuring the extinction coefficient of the Soret band (Falk, 1964). Ascans
RESULTS A bright red fluorescence, indicative of porphyrins, was evident only in extracts of perienteric fluid. Comparable results were obtained both for esters and free porphyrins, but because of the accuracy, speed and convenience of the extraction and separation methods, subsequent investigations were carried out with esterified porphyrins. When analysed by TLC, the extracts yielded a major and minor fluorescent band in the positions corresponding to protoporphyrin IX and coproporphyrin 111, respectively, as shown in Fig. I. Absorption spectra of the two substances were then determined and each possessed the characteristic spectrum of a porphyrin methyl ester. The spectrum of each substance was identical to the corresponding standard, both in wavelengths of the absorption maxima and ratios of extinction coefficients. Similar correspondence in spectra were noted when the esters were converted to their zinc and copper chelates (Doss, 1971). These criteria are sufficient (Marks, 1969) for establishing the identity of the major and minor bands as protoporphyrin IX and coproporphyrin III, respectively. No porphyrin esters were detected in any of the other tissues investigated, even when large quantities (up to 50 g in the case of muscle and reproductive tissues) were processed. Fluorescent material occasionally appeared in gut extracts, but was never noted in guts which had been flushed with saline prior to extraction. Thus, it is likely that any porphyrins present in the lumen of the worm gut are derived either from host gut contents or lumenal bacteria. In order to assess possible variability in porphyrin content of the perienteric fluid, worms of uniform size were divided into groups possessing either dark or light colored guts (Cain & Welshman, 1973), and porphyrins were esterified and extracted. As shown in Table 1, proto- and coproporphyrin concentrations in the fluid of worms with dark guts were nearly seven times greater than in worms with light guts, but the molar ratios of the two porphyrins
Origin
Fro. I. Fluorometric scan of TLC-separated porphyrin methyl esters from 250 ml of A. lumbricoides perienteric fluid (below) compared with a mixture of authentic standards containing 1 nmole of each porphyrin methyl ester.
were relatively constant in both groups. Similarly, the molar ratio of heme to protoporphyrin was nearly identical in the fluid of worms with light and dark guts. DISCUSSION There is little doubt, based on the spectrophotometric and chromatographic evidence presented here, that the free porphyrins of A. ~umbricoides perienteric fluid are coproporphyrin III and protoporphyrin IX. The most plausible alternatives might be the analogous Formyl porphyrins, which serve as prosthetic groups of cytochrome oxidase and of chlorocruorin, the hemoglobin of some annelids (Falk, 1964). In these porphyrins and their chromoproteins, however, the absorption spectra are shifted appreciably toward the longer wavelengths due to electron withdrawal by the formyl side chain (Sono & Asakura, 1974). No such phenomenon was observed in spectra of A. lumbricoides porphyrins. The source of the porphyrins in A. lumbricoides fluid is obscure, but at least three possibilities may be suggested. They may originate as breakdown products of endogenous heme proteins, although the presence of coproporphyrin makes this unlikely, since it would require an as yet unreported reversal of a portion of the heme synthesis pathway. The host gut contents represent a second possible source, but in this case, higher concentrations of copropor-
I.J.P. VOL. 6. 1976
Porphyrins
in Ascaris lumbrieoides
81
TABLE~-PORPHYRIN CONTENT OF Ascaris Iumbricoides PERIENTERIC FLUID Porphyrin concentration Proto Dark guts Light guts
4.08 (& 0.16)* 0.58 (i 0.044)
Molar ratios
Copro 0.29 (i 0.024) 0.028 (& 0.005)
Proto/Copro 21.47 20.71
Heme/Proto 4981 4996
* nmoles/lOO ml Perienteric fluid. Values represent average of 3 separate determinations
f S.D. phyrin might be expected, in keeping with reported ratios of free porphyrins in mammalian gut contents (Lemberg & Legge, 1949). Likewise, a dietary mechanism for obtaining porphyrins would have to be highly selective in order to account for the observed constancy in molar ratios of porphyrins to one another and to heme in worms containing dark or light colored guts. Selective dietary origin could account for the observed ratios if it operated together with a third possible mechanism, the accumulation of biosynthetic intermediates. The constancy of porphyrin : heme ratios supports this scheme, as does the presence of coproporphyrinogen oxidase (Cain, in press), the enzyme which converts coproporphyrinogen to protoporphyrinogen. The absence of uroporphyrin is inconsistent with the accumulation of intermediates, however, as is the limitation in the rate of iron incorporation into protoporphyrin, which would have to be invoked to explain the high levels of protoporphyrin. In other systems, aminolevulinic acid synthetase is known to be the ratelimiting enzyme in porphyrin synthesis (Granick & Urata, 1963). Clearly, an understanding of the source of porphyrins in A. hunbricoides requires more extensive testing of the above hypotheses. Free porphyrins are sporadically distributed throughout the animal kingdom, but relatively high concentrations occur only in species not ordinarily exposed to strong light, which is toxic to these organisms due to the photosensitizing effects of the porphyrins (Kennedy & Vevers, 1954; Kennedy, 1959). Burrowing annelids, e.g., contain appreciable concentrations of protoporphyrin (Mangum & Dales, 1965), as do benthic medusae (Herring, 1972) and certain nemertines (Fox & Vevers, 1960). Other porphyrins occur in animals of analogous habitats, e.g. uroporphyrin in planaria (MacRae, 1956, 1961, 1963) and in gastropod molluscs (Comfort, 1951), and coproporphyrin in millipedes (Needham, 1968). To this list should now be added A. lumbricoides which because of its dark habitat would not be expected to suffer deleterious effects from porphyrin accumulation. REFERENCES BOLLA R. I., WEINSTEINP. P. & Lou C. 1972. In vitro nutritional requirements of Nippostrongylus brasiliensis -1. Effects of sterols, sterol derivatives and heme compounds on the free-living stages. Comparative Biochemistry and 2hysiology 43B: 487-501.
BOLLA R. I., WEINSTEINP. P. & Lou C. 1974. In vitro nutritional requirements of Nippostrongyhts brasiliensis -11. Effects of heme compounds, porphyrins and bile pigments on the free-living stages. Comparative Biochemistry and Physiology 48B: 147-157. CAIN G. D. 1969. The source of hemoglobin in Philophthalmus megalurus and Fasciolopsis buski (Trematoda: Digenea). Journal of Parasitology 55: 307-310. CAIN G. D. (In Press) Coproporphyrinogen oxidase activity in Ascaris lumbriciodes eggs and muscle. Experimental
Parasitology.
CAIN G. D. & WELSHMANI. R. 1973. Effect of dietary tetrapyrroleson gut pigmentation and perienteric fluid hemoglobin concentration in Ascaris lumbricoides. International Journal for Parasitolopy 3: 623-630.
CHEAH K. S. 1968. The respirato;; components of Moniezia expansa (Cestoda). Biochimica et Biophysics Acta 153: 718-720.
CHEAHK. S. & CHANCEB. 1970. The oxidase system of Ascaris-muscle mitochondria. Biochimica et Biophysica Acta 223: 55-60.
CHEAH K. S. & PRICHARD R. K. 1975. The electron transport systems of Fasciola hepatica mitochondria. International Journal for Parasitology 5: 183-186. COMFORT A. 1951. The pigmentation of Molluscan shells. Biological Reviews Society 26: 285-301.
of
the
Cambridge
Philosophical
Doss M. 1967. The quantitative separation of porphyrins and protohaemin as methyl esters by thin-layer chromatography. Journal of Chromatography 3: 265-269.
Doss M. 1971. Conversion of porphyrin methyl esters to zinc and copper chelates for spectrophotometric analysis. Analytical Biochemistry 39: 7-14. Doss M., ULSHGFERB. & PHILLIP-DORMSTON W. K. 1971. Quantitative thin-layer chromatography of porphyrins by in situ fluorescence measurements. Journal of Chromatography 63: 113-120. DRESELE. I. B. & FALK J. E. 1956. Studies on the biosynthesis of blood pigments. 2. Haem and porphyrin formation in intact chicken erythrocytes. Biochemical Journal 63: 72-79.
FAIRBAIRND. 1970. Biochemical adaptation and loss of genetic capacity in helminth parasites. Biological Reviews 45: 29-72.
FALK J. E. 1961. Chromatography metalloporphyrins. Journal of
of porphyrins Chromatography
and 5:
277-299.
FALK J. E. 1964. Porphyrins and Metalloporphyrins, pp. 255. Elsevier, Amsterdam. Fox H. M. & VEVERSH. G. 1960. The Nature of Animal Colours, pp. 246. Sidgwick. & Jackson, London. GRANICKS. & URATA G. 1963. Increase in activity of 6aminolevulinic acid synthetase in liver mitochondria induced by feeding of 3,5-dicarbethoxy-1, 4-dihydrocollidine. Journal of Biological Chemistry 238: 821-827.
82
G. D. CAIN and FERN Bassow
HARPUR R. P. 1962. Maintenance of Ascaris lumbricoides z’rz vitro: a biochemical and statistical approach. Canadian Journal of Zoology 40: 991-1011. HERRINGP. J. 1972. Porphyrin pigmentation in deep-sea medusae. Nature, London 238: 276-277. HIEB W. F., STOKSTADE. L. R. & ROTHSTEINM. 1970. Heme requirement for reproduction of a free-living nematode. Science, New York 168: 143-144. HILL G. C., PERKOWSKIC. A. & MATHEWSONN. W. 1972. Purification and properties of cytochrome ~550 from Ascaris Iumbricoides. Biochimica et Biophysics Actu 236: 242-245. KENNEDY G. Y. 1959. A porphyrin pigment in the integument of Arion ater (L.). Jour~za/ of the Mor~~e Biological Association of the Utlited Kingdom 38: 27-32. KENNEDYG. Y. & VEVERSG. H. 1954. The occurrence of porphyrins in certain marine invertebrates. Journal of the Marine Biological Association of the United Kingdom 33: 663-676. LEE D. L. & SMITHM. H. 1965. Haemo8lobins of parasitic animals. Experimental Par~~tolo~y 16: 392-424. LEMBERGR. & LEGGEJ. W. 1949. Haematin Compounds and Bile Pigments, pp. 749. Interscience, New York. MACRAEE. K. 1956. The occurrence of porphyrin in the planarian. Biological Bulletin 110: 69--l& MACRAE E. K. 1961. Localization of porphyrin fluorescence in planarians. Science, Nets York 134: 331-332. MACRAEE. K. 1963. Chromatographic identification of porphyrins in several species of Turbellaria. Experientia 19: 77-78.
I.J.P. VOL.6. 1976
MANGUMC. P. & DALE.S R. P. 1956. Products of haemsynthesis in Polychaetes. Comparative Biochemistry and Physiology 15: 237-257. MARKS G. 1969. Heme and C’hloroohvll. Chemical. Biochemical and Medical Aspects, pp. 20s. Van No&and, London. NEEDHAMA. E. 1968. lntegumental pigments of the millipede, Polydesmus angustus f Latzel). Nature, London 217: 975-977. SANO S. & GRANICK S. 1961. Mitochondrial coproporphyrinogen oxidase and protoporphyrin formation. Jotrrnal of Bio/ogical Chemistry 236: 1 I73- I 180. SEIDELJ. S. 1971. Hemin as a requirement in the development in vitro of ~iytrlenolepi~ microstoma (Cestoda: Cyclophyliidea~. Jorcrrtaf of Parasitology 57: 566-570. SMITH M. H. & LEE D. L. 1963. Metabolism of haemoglobin and haematin compounds in Ascaris lumbricoides. Proceedings of the Royal Society (London) B 157: 234-257. SONOM. & ASAKURAT. 1974. Separation and properties of spirographis and isospirographis porphyrin dimethyl esters. Bjor~zemj~try 13: 4386-4394. TAIT G. H. 1968. General aspects of haem synthesis. In Biochemical Society Symposium 28 : Porphyrins and Related Cornpout& (Edited by GO~D~IN T. W.), pp. 19-34. Academic Press, New York. ULSH~~FER B. & Doss M. 1969. A sensitive method of documentation of porphyrin and hemin thin-layer chromatograms. Jourl~ai of Chromatograph~~ 44: 407-412. VON BRANDT. 1973. Biochemistry of Parasites. pp. 499. Academic Press, New York.
CAIN
FLUID
OF
and FERN BASSOW
of Zoology, The University of Iowa, Iowa City, Iowa 52242, U.S.A. (Received 23 April 1975)
Abstract-CAIN
G. D. and BASSOW F. 1976. Porphyrins in the perienteric fluid of Ascarfs ~~~~~~~c~~~e~. t~rern~~io~zul Journal fur Parusitafogy 6: 79-82. Porphyrins in the perienteric fluid of adult female A. lumhricoides were esterified in methanolic H,SOI, extracted in chloroform, separated by thin-layer chromatography, and identified spectrophotometrically before and after conversion to their zinc and copper chelates. Protoporphyrin IX was the major component, comprising 95.4% of the total; the remaining 4.6 ‘Awas coproporphyrin III. Uroporphyrin was not detected; no porphyrins were recovered from other worm tissues. Fluid from worms with light and dark colored guts varied in protoporphyrin content from 0.58 to 4.08 nmoles/ml, respectively, but fluid from both groups contained similar molar ratios of protoporphyrin, coproporphyr~n and heme. INDEX KEY WORDS: Ascaris lumbricoides; perienteric fluid; porphyrin; protoporphyrin IX; coproporphyrin III; thin-layer chromatography.
effect
in cultured
methyl ester;
that parasitic helminths lack the ability to synthesize porphyrin (Smith & Lee, 1963; Fairbairn, 1970; von Brand, 1973). It is therefore surprising that coproporphyrinogen oxidase, the penultimate enzyme in the porphyrin synthesis pathway (Sano & Granick, 1961; Tait, 1968) is evidently present at low activity levels in A. fumbr~coi~es mitochondria (Cain, in press). This finding suggested that the porphyrin pathway might be at least partially operative and prompted a search for the biosynthetic intermediates, uro-, copro- and protoporphyrin in A. lumbricoides.
IRON-PORPHYRIN complexes are common constituents of parasitic helminths, where they are found both in hemoglobins (for review, see Lee & Smith, 1965) and cytochromes (Cheah & Chance, 1970; Cheah, 1968; Hill, Perkowski & Mathewson, 1972; Cheah & Pritchard, 1975). Despite the wide occurrence of heme proteins in helminths, however, very little is known about the origin and metabolism of heme and porphyrin in these organisms. Nutritional studies with nematodes suggest that heme and porphyrins have a growth-promoting effect in Nippostr~ngy~us brasiliensis (Bolla, Weinstein & Lou, 1972, 1974), and in a free-living species, ~~en~rt~abdit~~briggsae (Hieb, Stokstad & Rothstein, 1970). The nature of the effect is poorly understood, but in at least one species, Ascaris lumbricoides, administration of hemes and certain other porphyrins and metalloporphyrins under in viva (Cain & Welshman, 1973) and in vitro (Smith & Lee, 1963) conditions causes an increase in hemoglobin production. In parasitic flatworms, heme has a growth stimulating
porphyrin
MATERIALS
AND METHODS
Adult female A. ~~mbric~jdeswere obtained at a local slaughterhouse and immediately transported to the laboratory. Worms were washed thoroughly in Harpur’s saline solution (Harpur, 1962) and dissected carefully so as not to contaminate tissues with worm gut contents. Muscle and reproductive tissues were washed in three changes of saline; guts were washed free of contents by flushing the lumens exhaustively with saline by means of a Pasteur pipette. Porphyrins in lyophilized tissues and perienteric Ruid were either extracted as free porphyrins using the method of Dresel & Falk (1956) or esterified with methanolic H,SO$ and extracted according to the methods of Doss (1971). Free porphyrins were separated by paper chromatography (Falk, 1961) and porphyrin methyl esters by thin-layer chromatography (TLC) on silica gel H (Doss, 1967). The TLC plates were scanned on a Turner model 111 fluorometer as described by Doss, Ulshofer & PhillipDormston (1971) and then photographed (Ulshofer & Doss, 1969). Quantification of porphyrin esters by fluorometric scanning (Doss et al., 1971) was not per-
Hymertolepis microstoma
(Seidel, 19711, even though this tapeworm apparently does not contain hemoglobin. Nutritional studies on trematodes are lacking, but the heme precursor, Saminolevulinic acid, was not incorporated into hemoglobins of Fascioiopsis buski and Philophthalmus megalurus under conditions where Iabelled amino acids were heavily incorporated into the protein moiety (Cain, 1969). The above results are usually interpreted to mean 79
80
G. D. CAIN and FERN BASSOW
formed, since fatty acid methyl esters, abundant in worm samples, tended to cochromatograph with protoporphyrin dimethyl ester and interfered with its fluorescence. Fluorescent porphyrin esters corresponding in chromatographic mobility to authentic standards (Sigma Chemical Co., St. Louis, MO.) were scraped from TLC plates and eluted from the silica gel with chloroform, while free porphyrin spots were eluted with 1 N-HCI following paper chromatography. Eluted substances were scanned in a Beckman Acta III spectrophotometer and quantified as free porphyrins and methyl esters (Falk, 1964) and as zinc and copper chelates of the methyl esters (Doss, 1971). Heme dimethyl ester, which remained at the origin under the TLC conditions em-
I.J.P. VOL.6. 1976
URO III
COPRO III
PROTOIX
ployed, was eluted from the gel with ethyl acetate and quantified, after appropriate dilution, by measuring the extinction coefficient of the Soret band (Falk, 1964). Ascans
RESULTS A bright red fluorescence, indicative of porphyrins, was evident only in extracts of perienteric fluid. Comparable results were obtained both for esters and free porphyrins, but because of the accuracy, speed and convenience of the extraction and separation methods, subsequent investigations were carried out with esterified porphyrins. When analysed by TLC, the extracts yielded a major and minor fluorescent band in the positions corresponding to protoporphyrin IX and coproporphyrin 111, respectively, as shown in Fig. I. Absorption spectra of the two substances were then determined and each possessed the characteristic spectrum of a porphyrin methyl ester. The spectrum of each substance was identical to the corresponding standard, both in wavelengths of the absorption maxima and ratios of extinction coefficients. Similar correspondence in spectra were noted when the esters were converted to their zinc and copper chelates (Doss, 1971). These criteria are sufficient (Marks, 1969) for establishing the identity of the major and minor bands as protoporphyrin IX and coproporphyrin III, respectively. No porphyrin esters were detected in any of the other tissues investigated, even when large quantities (up to 50 g in the case of muscle and reproductive tissues) were processed. Fluorescent material occasionally appeared in gut extracts, but was never noted in guts which had been flushed with saline prior to extraction. Thus, it is likely that any porphyrins present in the lumen of the worm gut are derived either from host gut contents or lumenal bacteria. In order to assess possible variability in porphyrin content of the perienteric fluid, worms of uniform size were divided into groups possessing either dark or light colored guts (Cain & Welshman, 1973), and porphyrins were esterified and extracted. As shown in Table 1, proto- and coproporphyrin concentrations in the fluid of worms with dark guts were nearly seven times greater than in worms with light guts, but the molar ratios of the two porphyrins
Origin
Fro. I. Fluorometric scan of TLC-separated porphyrin methyl esters from 250 ml of A. lumbricoides perienteric fluid (below) compared with a mixture of authentic standards containing 1 nmole of each porphyrin methyl ester.
were relatively constant in both groups. Similarly, the molar ratio of heme to protoporphyrin was nearly identical in the fluid of worms with light and dark guts. DISCUSSION There is little doubt, based on the spectrophotometric and chromatographic evidence presented here, that the free porphyrins of A. ~umbricoides perienteric fluid are coproporphyrin III and protoporphyrin IX. The most plausible alternatives might be the analogous Formyl porphyrins, which serve as prosthetic groups of cytochrome oxidase and of chlorocruorin, the hemoglobin of some annelids (Falk, 1964). In these porphyrins and their chromoproteins, however, the absorption spectra are shifted appreciably toward the longer wavelengths due to electron withdrawal by the formyl side chain (Sono & Asakura, 1974). No such phenomenon was observed in spectra of A. lumbricoides porphyrins. The source of the porphyrins in A. lumbricoides fluid is obscure, but at least three possibilities may be suggested. They may originate as breakdown products of endogenous heme proteins, although the presence of coproporphyrin makes this unlikely, since it would require an as yet unreported reversal of a portion of the heme synthesis pathway. The host gut contents represent a second possible source, but in this case, higher concentrations of copropor-
I.J.P. VOL. 6. 1976
Porphyrins
in Ascaris lumbrieoides
81
TABLE~-PORPHYRIN CONTENT OF Ascaris Iumbricoides PERIENTERIC FLUID Porphyrin concentration Proto Dark guts Light guts
4.08 (& 0.16)* 0.58 (i 0.044)
Molar ratios
Copro 0.29 (i 0.024) 0.028 (& 0.005)
Proto/Copro 21.47 20.71
Heme/Proto 4981 4996
* nmoles/lOO ml Perienteric fluid. Values represent average of 3 separate determinations
f S.D. phyrin might be expected, in keeping with reported ratios of free porphyrins in mammalian gut contents (Lemberg & Legge, 1949). Likewise, a dietary mechanism for obtaining porphyrins would have to be highly selective in order to account for the observed constancy in molar ratios of porphyrins to one another and to heme in worms containing dark or light colored guts. Selective dietary origin could account for the observed ratios if it operated together with a third possible mechanism, the accumulation of biosynthetic intermediates. The constancy of porphyrin : heme ratios supports this scheme, as does the presence of coproporphyrinogen oxidase (Cain, in press), the enzyme which converts coproporphyrinogen to protoporphyrinogen. The absence of uroporphyrin is inconsistent with the accumulation of intermediates, however, as is the limitation in the rate of iron incorporation into protoporphyrin, which would have to be invoked to explain the high levels of protoporphyrin. In other systems, aminolevulinic acid synthetase is known to be the ratelimiting enzyme in porphyrin synthesis (Granick & Urata, 1963). Clearly, an understanding of the source of porphyrins in A. hunbricoides requires more extensive testing of the above hypotheses. Free porphyrins are sporadically distributed throughout the animal kingdom, but relatively high concentrations occur only in species not ordinarily exposed to strong light, which is toxic to these organisms due to the photosensitizing effects of the porphyrins (Kennedy & Vevers, 1954; Kennedy, 1959). Burrowing annelids, e.g., contain appreciable concentrations of protoporphyrin (Mangum & Dales, 1965), as do benthic medusae (Herring, 1972) and certain nemertines (Fox & Vevers, 1960). Other porphyrins occur in animals of analogous habitats, e.g. uroporphyrin in planaria (MacRae, 1956, 1961, 1963) and in gastropod molluscs (Comfort, 1951), and coproporphyrin in millipedes (Needham, 1968). To this list should now be added A. lumbricoides which because of its dark habitat would not be expected to suffer deleterious effects from porphyrin accumulation. REFERENCES BOLLA R. I., WEINSTEINP. P. & Lou C. 1972. In vitro nutritional requirements of Nippostrongylus brasiliensis -1. Effects of sterols, sterol derivatives and heme compounds on the free-living stages. Comparative Biochemistry and 2hysiology 43B: 487-501.
BOLLA R. I., WEINSTEINP. P. & Lou C. 1974. In vitro nutritional requirements of Nippostrongyhts brasiliensis -11. Effects of heme compounds, porphyrins and bile pigments on the free-living stages. Comparative Biochemistry and Physiology 48B: 147-157. CAIN G. D. 1969. The source of hemoglobin in Philophthalmus megalurus and Fasciolopsis buski (Trematoda: Digenea). Journal of Parasitology 55: 307-310. CAIN G. D. (In Press) Coproporphyrinogen oxidase activity in Ascaris lumbriciodes eggs and muscle. Experimental
Parasitology.
CAIN G. D. & WELSHMANI. R. 1973. Effect of dietary tetrapyrroleson gut pigmentation and perienteric fluid hemoglobin concentration in Ascaris lumbricoides. International Journal for Parasitolopy 3: 623-630.
CHEAH K. S. 1968. The respirato;; components of Moniezia expansa (Cestoda). Biochimica et Biophysics Acta 153: 718-720.
CHEAHK. S. & CHANCEB. 1970. The oxidase system of Ascaris-muscle mitochondria. Biochimica et Biophysica Acta 223: 55-60.
CHEAH K. S. & PRICHARD R. K. 1975. The electron transport systems of Fasciola hepatica mitochondria. International Journal for Parasitology 5: 183-186. COMFORT A. 1951. The pigmentation of Molluscan shells. Biological Reviews Society 26: 285-301.
of
the
Cambridge
Philosophical
Doss M. 1967. The quantitative separation of porphyrins and protohaemin as methyl esters by thin-layer chromatography. Journal of Chromatography 3: 265-269.
Doss M. 1971. Conversion of porphyrin methyl esters to zinc and copper chelates for spectrophotometric analysis. Analytical Biochemistry 39: 7-14. Doss M., ULSHGFERB. & PHILLIP-DORMSTON W. K. 1971. Quantitative thin-layer chromatography of porphyrins by in situ fluorescence measurements. Journal of Chromatography 63: 113-120. DRESELE. I. B. & FALK J. E. 1956. Studies on the biosynthesis of blood pigments. 2. Haem and porphyrin formation in intact chicken erythrocytes. Biochemical Journal 63: 72-79.
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