- Email: [email protected]
A preliminary study of fungal melanin by infrared spectroscopy
Geoderma, 24 (1980) 207--213
207
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
A PRELIMINARY STUDY OF FUNGAL MELANIN BY INFRARED SPECTROSCOPY
J.D. RUSSELL, D. JONES, D. VAUGHAN and A.R. FRASER
Departments of Spectrochemistry, Microbiology and Soil Organic Chemistry, Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen, AB9 2QJ (Great Britain) (Received September 3, 1979; January 25, 1980)
ABSTRACT Russell, J.D., Jones, D., Vaughan, D. and Fraser, A.R., 1980. A preliminary study of fungal melanin by infrared spectroscopy. Geoderma, 24: 207--213. Infrared spectra of melanin and methylated melanin have been interpreted to show the existence of carboxyl groups in two different environments in the melanin structure. In one, there are a small number of carboxyl groups with an absorption band at about 1700 cm-1; these are relatively free and are capable of interaction with alkali to produce carboxylate in the normal way. In the other environment, the carboxyl groups, absorbing at 1615 cm -1, are conjugated and strongly hydrogen bonded to presumably phenolic hydroxyl groups, and cannot be neutralized with alkali. They do, however, react with diazomethane, as do the free carboxyls, to produce methyl ester absorbing at 1728 cm -1. The significance of these conclusions for the survival of melanin in soil and its reactions therein, is briefly discussed.
INTRODUCTION
Melanins occur widely in nature in plant, animal and microbial tissue as melanoproteins (Nicolaus and Piattelli, 1965). They are extractable as such by water or alkali, but cleavage from the protein moiety renders the melanin insoluble in all solvents (Blois, 1978). This splitting is likely to occur enzymatically in soil, the melanin being only slowly metabolized further (Linhares and Martin, 1978). Melanins are therefore likely to persist in soil, but little is known about their structure or their contribution to soil organic matter and its reactions. To try to gain some information about the structure of this intractible biopolymer, a preliminary investigation of the melanin from the fungus Sclerotinia sclerotiorum described by Jones (1970) has been made using infrared spectroscopy, methylation and chemical analysis. 0016-7061/80/0000--0000/$ 02.25 © 1980 Elsevier Scientific Publishing Company
208
MATERIALS AND METHODS Sclerotia were obtained from cultures of Sclerotinia sclerotiorum (Lib.) de Bary, grown on a medium consisting of 4% (w/v) Oxoid malt extract and 1.2% (w/v) Oxoid No. 3 agar, and incubated for 2--3 weeks at 20°C (Jones, 1970).
Extraction of pigment Two procedures were used: (1) Whole air-dried sclerotia were ground to a fine powder using a mortar and pestle. These sclerotia may have autolysed to some extent. The powder, 50 mg, was boiled in 10 ml 0.5 M NaOH for 3 h giving a dark b r o w n suspension which was centrifuged at 2000 g for 10 min. The debris, which was still pigmented, was discarded. The brown alkaline centrifugate was brought to pH 2.0 with 6 M HC1 and the precipitated pigment washed with distilled water and dried at 50 ° C. (2) The black rind containing the melanin pigment was removed from the sclerotia with a razor blade and washed in water. To remove the polysaccharide and protein, the rind from 200 sclerotia was boiled in 10 ml 1.5 M HC1 for 1.5 h, washed twice in 10 ml water, then boiled in 10 ml 5.4 M KOH for 1.5 h, and washed twice in 10 ml water. To remove chitin, the residue was stirred in 10 ml 8 M HC1 at 2°C for 2 h, washed four times in 10 ml water and finally dried at 50°C. The yield was 35 mg. To remove residual protein, the pigments isolated b y these methods were hydrolysed with boiling 6 M HC1 for several hours. The insoluble residues were washed with water and dried at 50 ° C. Pigments treated in this way are in the acid form. Salt forms were prepared b y subsequent treatment with NaOH followed b y water washing.
Methylation of hydrolysed pigment The pigment from procedure 2 was suspended in 2 ml re-distilled diethyl ether and methylated b y distilling ethereal diazomethane (prepared b y adding 5 g nitrosomethylurea to a mixture of 10 ml 9 M KOH and 10 ml re-distilled diethyl ether) into the suspension until the yellow colour of the reagent persisted. The ether was allowed to evaporate at r o o m temperature. The sample was remethylated a further 5 times, then washed successively with chloroform, ether and water. Finally it was treated with 5% HF to remove silica which appears in .some methylated products.
Chemical analysis The total nitrogen contents of the pigments were measured in H2SO4 digests b y the m e t h o d of Searcy et al. (1967). The inorganic-P content in the acid digest was measured b y the method of Allen (1940). The m e t h o x y l content
209
of the methylated samples was measured by the Zeisel method (Belcher and Godbert, 1954).
Infrared spectrometry I.R. spectra of samples (0.8 mg in 13 mm diameter KBr pressed disks) were recorded on a Perkin-Elmer 577 spectrometer over the range 4000-200 cm -1. Disks were heated at 150°C for 16 h to remove adsorbed water. RESULTS AND DISCUSSION
Alkaline extraction of the pigment from the ground sclerotia, followed by precipitation with acid (method 1), yields a melanoprotein whose spectrum (Fig. la) is characterized by the intense secondary amide absorption bands of protein at 1660 and 1520 cm -1. Similar protein-dominated spectra have been obtained for so called fungal melanins (Filip et al., 1974) and for bacterial 'humic acids' (Kosinkiewicz, 1978). It is suggested that the use of these terminologies is unnecessarily misleading, and that the more correct melanoI
-- I
Icn ?2
J-
I
~
I
I
]
I-
\ ~ _ _ ~ .
I
I
co 2
1660
2925
1615
1 4o-
L
L
30
I
I
20
I
6
1 L
16
5 I
~ I
12
''~'F~ I
L
8
c J
I
4
V/~oocm -~
Fig. 1. I n f r a r e d s p e c t r a o f h e a t e d K B r d i s k s o f s c l e r o t i n i a p i g m e n t : a. i s o l a t e d b y p r o c e d u r e 1 in m a t e r i a l s a n d m e t h o d s ; b. as a, h y d r o l y s e d w i t h b o i l i n g 6 M HCI; c. as b, t r e a t e d with NaOH then water washed.
210
protein should be used for all alkali-soluble biological pigments of this type: melanin, as defined by Blois (1978), should be reserved solely for the alkaliinsoluble pigment which does not contain protein. Acid hydrolysis of the sclerotial melanoprotein yields an alkali-insoluble melanin which, from the absence of the 1660 and 1520 cm -1 secondary amide bands in its spectrum (Fig. lb), contains little protein. The dominant absorption bands at 1615 cm -1 and 1240 cm-', with weaker bands at 3450, 1700 and 830 cm-' and broad unresolved absorption in the range 3300--2400 cm -~ are all typical of melanin (Jones, 1970), but the spectrum contains stronger C-H absorption bands at 2850--2960 cm -1 and 1450 cm -~, stronger C = O absorption at 1700 cm -1, and weak bands of unknown origin at 950, 750 and 700 cm -~. A preparation lacking the component(s) responsible for the last three absorption bands was obtained from extraction procedure 2. The initial HC1 treatment used in this method, however, hydrolysed the native melanoprotein, rendering the melanin insoluble in the subsequent alkali treatment. Further purification was then only possible by removing nonpigmented wall components such as polysaccharide and chitin. The IR spectrum of the melanin prepared by this method (Fig. 2a) is the same as that shown by Jones {1970). The absorption pattern r
I
i co 2
i
f
I
i
" i
r
1390
1240
1
r
]
1615
b
1615 1585
1728 16
1 40
30
J
I 20
I
J 16
/ 1030 1260 1085
J
P
C
[
I
12
8 V/oocm
~
J 4
-I
F i g . 2. I n f r a r e d s p e c t r a o f h e a t e d K B r d i s k s o f sclerotinia pigment: a. i s o l a t e d b y p r o c e d u r e 2 in materials and m e t h o d s ; b. a s a, t r e a t e d w i t h N a O H then water w a s h e d ; c. a s a, m e t h y lated 6 times with diazomethane.
211
TABLE I Chemical C o m p o s i t i o n (%) o f Melanins f r o m S. Sclerotiorum and Ustilago rnaydis .1 Melanin s o u r c e
C
H
N
P
OMe
Ash
S. sclerotiorum
60.3
3.7
1.2 0.9
0.06 0.04
--12.6
0.6 _,2 _,2
62.8
3.4
1.0
HCI h y d r o l . CH=N 2 m e t h y l a t e d
Ustilago maydis CH2N 2 m e t h y l a t e d
12.2
,1 Nicolaus and Piattelli, 1965. *~ I n s u f f i c i e n t sample for ash d e t e r m i n a t i o n .
is virtually unchanged on further HC1 hydrolysis of the pigment, its N content decreasing from 1.2 to 0.9% (table I). Such a low nitrogen content suggests that the origin of the melanin, as proposed b y Jones (1970), is probably catechol rather than DOPA, thus classifying the melanin with those of vegetable origin (Nicolaus and Piattelli, 1965). Indeed, the spectrum of the sclerotinia melanin resembles that of a synthetic pigment prepared b y the enzymic oxidation of a mixture of simple phenols including catechol (Filip et al., 1974) and of catechol alone (Andrews and Pridham, 1967). The similarity is only superficial, however, in that the 1615 cm-' band is weaker than that in the 1200--1300 cm -1 region for the synthetic polymers whereas in melanin the 1615 cm -1 band is stronger (Figs. l b , 2a), and unlike natural melanins, these synthetic polymers are soluble in alkali. The above classification of the sclerotinia melanin as a vegetable pigment is apparently at variance with the findings of Liu et al. (1977) who showed that the pigment from the sclerotia of Sclerotium rolfsii Sacc. contained up to 8% N and yielded indole on alkaline fusion, suggesting that their melanin was of animal origin. However, its large N content and solubility at b o t h high and low pH indicates that it is most probably a melanoprotein (Blois, 1978). The spectra of melanin isolated from acidic media (Figs. l b , 2a) differ considerably from those of the melanin treated with alkali (Figs. lc, 2b). The band at 1700 cm -t disappears, broad absorption at 3300--2400 cm -1 and the band at 1240 cm -~ b e c o m e weaker, and simultaneously a new band appears at 1585 cm -~ and absorption at 1390 cm -~ becomes stronger. These changes are consistent with the conversion of a small proportion of free carboxylic acid groups to carboxylate b y alkali, b u t it is significant that the principal band at 1615 cm -1 is n o t affected b y this treatment, and that considerable intensity still remains at 1240 cm -~ and in the 3300--2400 cm -~ region. To try to identify the origin of these three features, the melanin was repeatedly methylated with diazomethane to give a product with a m e t h o x y l content (12.6%) close to that obtained b y Nicolaus and Piattelli (1965) for methylated melanin from the spores of Ustilago maydis (Table I). The IR spectrum of the methylated sclerotinia melanin (Fig. 2c) shows considerable
212 weakening of b o t h the 1615 cm -1 band and of absorption at 3300--2400 cm -1, a shift of the 1240 cm-' band to 1260 cm-' and the appearance of a new band at 1728 cm -1, spectra run at intermediate stages of methylation confirming that the enhancement at 1728 cm -1 occurred at the expense of the 1615 cm -1 band. The 1260 cm -1 and 1728 cm -~ bands arise from esters, possibly unsaturated or aryl (Bellamy, 1975), leading to the conclusion that the 1615 cm-' band arises from carboxylic acid groups. An alternative assignment of the 1615 cm-' band to carboxylate groups is untenable because of the very small ash content of the pigment (Table I). Ester groups derived from the small number of free carboxylic acid groups absorbing at 1700 cm -~ will also contribute to the 1728 cm-' band. The 1615 cm -1 band lies outside the range of even internally hydrogen-bonded carboxylic acid groups in simple structures (Bellamy, 1975}, but in complex, more highly oxidized systems, such factors as extended conjugation and hydrogen bonding to phenolic OH groups might combine to produce these low frequencies. Certainly, the presence of such groups is strongly indicated b y the loss of absorption between 3500 and 3000 cm -1 in Fig. 2a on salt formation (Fig. 2b), and on methylation (Fig. 2c), and particularly b y the appearance in the spectrum of methylated melanin (Fig. 2c) of new bands in the 1200--1020 cm -1 region which is characteristic for alkylarylether groups. The formation of a methyl ester which led to the assignment of the 1615 cm -~ band to conjugated, strongly hydrogen b o n d e d carboxyl groups invalidates previous assignments of this band in melanin spectra solely to conjugated carbonyl groups (Bonnet and Duncan, 1962) or to chelated quinones (Andrews and Pridham, 1967) because methylation o f such systems would not give rise to a group absorbing at a frequency as high as 1728 cm -1. For the same reason, an assignment to/~-diketones, proposed b y Theng and Posner (1967) for an analogous band at 1610 cm-' in humic acid, is precluded, as is an assignment to graphitic structures suggested b y Friedel and Carlson (1972) for a similar band in coal spectra. The results presented here suggest that the melanin structure might be similar to the oxidized polycyclic catechol p o l y m e r proposed b y Nicolaus and Piattelli (1965) in which phenolic OH and carboxylic acid groups occur. The majority of both groups are mutually involved in strong hydrogen bonds, the remainder being relatively free. It is this latter t y p e which participates in salt formation, a property studied b y Bruenger et al. (1967) and more recently b y Larsson and Tjalve (1978) who concluded that melanin behaves as a weak cation exchanger. In soil, melanin will therefore make a significant contribution to the movement and availability of metal ions in the soil through these readily available carboxyl groups. The strongly hydrogen bonded carboxyl groups are unable to participate in this reaction, b u t the observation that diazomethane can break this b o n d and produce ester and m e t h o x y l groups suggests that these carboxyl groups m a y n o t be permanently unavailable. Indeed, Linhares and Martin (1978), while showing that, in soil, fungal melanins were almost as resistant to biodegradation as humic acids, nevertheless reported a loss of up to 15% of added '4C in 12 weeks, indicating structural decomposition which might
213
alter the hydrogen bond status of the carboxyl groups making them more available in ion exchange reactions. The possible contribution of melanins to the humic fraction o f soil organic matter is currently being actively investigated.
REFERENCES Allen, R.J.L., 1940. The estimation of phosphorus. Biochem. J., 34: 858--865. Andrews, R.S. and Pridham, J.B., 1967. Melanins from DOPA-containing plants. Phytochemistry, 6: 13--18. Belcher, R. and Godbert, A.L., 1954. In: Semi-Micro Quantitative Analysis. Longmans, Green and Co., London, 2rid ed., pp. 155--159. Bellamy, L.J., 1975. The Infrared Spectra of Complex Molecules, 1, Chapman and Hall, London, 3rd ed. Blois, M.S., 1978. The melanins: their synthesis and structure. Photochem. Photobiol. Rev., 3: 115--134. Bonnet, T.G. and Duncan, A., 1962. Infrared spectra of some melanins. Nature, 194: 1078--1079. Bruenger, F.W., Stover, B.J. and Atherton, D.R., 1967. The incorporation of various metal ions into in vivo and in vitro melanin. Radiat. Res., 32: 1--12. Filip, Z., Haider, K., Beutelspacher, H. and Martin, J.P., 1974. Comparisons of IR-spectra from melanins of microscopic soil fungi, humic acids and model phenol polymers. Geoderma, 11: 37--52. Friedel, R.A. and Carlson, G.L., 1972. Difficult carbonaceous materials and their infrared and Raman spectra. Reassignments for coal spectra. Fuel, 51: 194--198. Jones, D., 1970. Ultrastructure and composition of the cell-walls of Sclerotinia sclerotiorum. Trans. Br. Mycol. Soc., 54: 351--360. Kosinkiewicz, B., 1977. Humic-like substances of bacterial origin, I. Some aspects of the formation and nature of humic-like substances produced by Pseudomonas. Acta Microbiol. Pol., 26: 377--386. Larsson, B. and Tjalve, H., 1978. Studies on the melanin affinity of metal ions. Acta Physiol. Scand., 104: 479--484. Linhares, L.F. and Martin, J.P., 1978. Decomposition in soil of the humic acid-type polymers (melanins) of Eurotrium echinulaturn, Aspergillus glaucus sp. and other fungi. Soil Sci. Soc. Am. J., 42:738--743. Liu, T.M.E., Yu, P.H. and Wu, L.C., 1977. Isolation purification and identification of dark pigments in the sclerotia of Sclerotiurn rolfsii Sacc. Chih Wu Pao Hu Hsueh Hui Hui K'an, 19: 223--237. Nicolaus, R.A. and Piattelli, M., 1965. Progress in the chemistry of natural black pigments. Rend. Acad. Sci. Fis. Mat., 32: 83--97. Searcy, R.L., Reardon, J.E. and Foreman, J.A., 1967. A new photometric method for serum urea nitrogen determination. Am. J. Med. Technol., 33: 1---6. Theng, B.K.G. and Posner, A.M., 1967. Nature of the carbonyl groups in soil humic acid. Soil Sci., 104: 191--201.
207
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
A PRELIMINARY STUDY OF FUNGAL MELANIN BY INFRARED SPECTROSCOPY
J.D. RUSSELL, D. JONES, D. VAUGHAN and A.R. FRASER
Departments of Spectrochemistry, Microbiology and Soil Organic Chemistry, Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen, AB9 2QJ (Great Britain) (Received September 3, 1979; January 25, 1980)
ABSTRACT Russell, J.D., Jones, D., Vaughan, D. and Fraser, A.R., 1980. A preliminary study of fungal melanin by infrared spectroscopy. Geoderma, 24: 207--213. Infrared spectra of melanin and methylated melanin have been interpreted to show the existence of carboxyl groups in two different environments in the melanin structure. In one, there are a small number of carboxyl groups with an absorption band at about 1700 cm-1; these are relatively free and are capable of interaction with alkali to produce carboxylate in the normal way. In the other environment, the carboxyl groups, absorbing at 1615 cm -1, are conjugated and strongly hydrogen bonded to presumably phenolic hydroxyl groups, and cannot be neutralized with alkali. They do, however, react with diazomethane, as do the free carboxyls, to produce methyl ester absorbing at 1728 cm -1. The significance of these conclusions for the survival of melanin in soil and its reactions therein, is briefly discussed.
INTRODUCTION
Melanins occur widely in nature in plant, animal and microbial tissue as melanoproteins (Nicolaus and Piattelli, 1965). They are extractable as such by water or alkali, but cleavage from the protein moiety renders the melanin insoluble in all solvents (Blois, 1978). This splitting is likely to occur enzymatically in soil, the melanin being only slowly metabolized further (Linhares and Martin, 1978). Melanins are therefore likely to persist in soil, but little is known about their structure or their contribution to soil organic matter and its reactions. To try to gain some information about the structure of this intractible biopolymer, a preliminary investigation of the melanin from the fungus Sclerotinia sclerotiorum described by Jones (1970) has been made using infrared spectroscopy, methylation and chemical analysis. 0016-7061/80/0000--0000/$ 02.25 © 1980 Elsevier Scientific Publishing Company
208
MATERIALS AND METHODS Sclerotia were obtained from cultures of Sclerotinia sclerotiorum (Lib.) de Bary, grown on a medium consisting of 4% (w/v) Oxoid malt extract and 1.2% (w/v) Oxoid No. 3 agar, and incubated for 2--3 weeks at 20°C (Jones, 1970).
Extraction of pigment Two procedures were used: (1) Whole air-dried sclerotia were ground to a fine powder using a mortar and pestle. These sclerotia may have autolysed to some extent. The powder, 50 mg, was boiled in 10 ml 0.5 M NaOH for 3 h giving a dark b r o w n suspension which was centrifuged at 2000 g for 10 min. The debris, which was still pigmented, was discarded. The brown alkaline centrifugate was brought to pH 2.0 with 6 M HC1 and the precipitated pigment washed with distilled water and dried at 50 ° C. (2) The black rind containing the melanin pigment was removed from the sclerotia with a razor blade and washed in water. To remove the polysaccharide and protein, the rind from 200 sclerotia was boiled in 10 ml 1.5 M HC1 for 1.5 h, washed twice in 10 ml water, then boiled in 10 ml 5.4 M KOH for 1.5 h, and washed twice in 10 ml water. To remove chitin, the residue was stirred in 10 ml 8 M HC1 at 2°C for 2 h, washed four times in 10 ml water and finally dried at 50°C. The yield was 35 mg. To remove residual protein, the pigments isolated b y these methods were hydrolysed with boiling 6 M HC1 for several hours. The insoluble residues were washed with water and dried at 50 ° C. Pigments treated in this way are in the acid form. Salt forms were prepared b y subsequent treatment with NaOH followed b y water washing.
Methylation of hydrolysed pigment The pigment from procedure 2 was suspended in 2 ml re-distilled diethyl ether and methylated b y distilling ethereal diazomethane (prepared b y adding 5 g nitrosomethylurea to a mixture of 10 ml 9 M KOH and 10 ml re-distilled diethyl ether) into the suspension until the yellow colour of the reagent persisted. The ether was allowed to evaporate at r o o m temperature. The sample was remethylated a further 5 times, then washed successively with chloroform, ether and water. Finally it was treated with 5% HF to remove silica which appears in .some methylated products.
Chemical analysis The total nitrogen contents of the pigments were measured in H2SO4 digests b y the m e t h o d of Searcy et al. (1967). The inorganic-P content in the acid digest was measured b y the method of Allen (1940). The m e t h o x y l content
209
of the methylated samples was measured by the Zeisel method (Belcher and Godbert, 1954).
Infrared spectrometry I.R. spectra of samples (0.8 mg in 13 mm diameter KBr pressed disks) were recorded on a Perkin-Elmer 577 spectrometer over the range 4000-200 cm -1. Disks were heated at 150°C for 16 h to remove adsorbed water. RESULTS AND DISCUSSION
Alkaline extraction of the pigment from the ground sclerotia, followed by precipitation with acid (method 1), yields a melanoprotein whose spectrum (Fig. la) is characterized by the intense secondary amide absorption bands of protein at 1660 and 1520 cm -1. Similar protein-dominated spectra have been obtained for so called fungal melanins (Filip et al., 1974) and for bacterial 'humic acids' (Kosinkiewicz, 1978). It is suggested that the use of these terminologies is unnecessarily misleading, and that the more correct melanoI
-- I
Icn ?2
J-
I
~
I
I
]
I-
\ ~ _ _ ~ .
I
I
co 2
1660
2925
1615
1 4o-
L
L
30
I
I
20
I
6
1 L
16
5 I
~ I
12
''~'F~ I
L
8
c J
I
4
V/~oocm -~
Fig. 1. I n f r a r e d s p e c t r a o f h e a t e d K B r d i s k s o f s c l e r o t i n i a p i g m e n t : a. i s o l a t e d b y p r o c e d u r e 1 in m a t e r i a l s a n d m e t h o d s ; b. as a, h y d r o l y s e d w i t h b o i l i n g 6 M HCI; c. as b, t r e a t e d with NaOH then water washed.
210
protein should be used for all alkali-soluble biological pigments of this type: melanin, as defined by Blois (1978), should be reserved solely for the alkaliinsoluble pigment which does not contain protein. Acid hydrolysis of the sclerotial melanoprotein yields an alkali-insoluble melanin which, from the absence of the 1660 and 1520 cm -1 secondary amide bands in its spectrum (Fig. lb), contains little protein. The dominant absorption bands at 1615 cm -1 and 1240 cm-', with weaker bands at 3450, 1700 and 830 cm-' and broad unresolved absorption in the range 3300--2400 cm -~ are all typical of melanin (Jones, 1970), but the spectrum contains stronger C-H absorption bands at 2850--2960 cm -1 and 1450 cm -~, stronger C = O absorption at 1700 cm -1, and weak bands of unknown origin at 950, 750 and 700 cm -~. A preparation lacking the component(s) responsible for the last three absorption bands was obtained from extraction procedure 2. The initial HC1 treatment used in this method, however, hydrolysed the native melanoprotein, rendering the melanin insoluble in the subsequent alkali treatment. Further purification was then only possible by removing nonpigmented wall components such as polysaccharide and chitin. The IR spectrum of the melanin prepared by this method (Fig. 2a) is the same as that shown by Jones {1970). The absorption pattern r
I
i co 2
i
f
I
i
" i
r
1390
1240
1
r
]
1615
b
1615 1585
1728 16
1 40
30
J
I 20
I
J 16
/ 1030 1260 1085
J
P
C
[
I
12
8 V/oocm
~
J 4
-I
F i g . 2. I n f r a r e d s p e c t r a o f h e a t e d K B r d i s k s o f sclerotinia pigment: a. i s o l a t e d b y p r o c e d u r e 2 in materials and m e t h o d s ; b. a s a, t r e a t e d w i t h N a O H then water w a s h e d ; c. a s a, m e t h y lated 6 times with diazomethane.
211
TABLE I Chemical C o m p o s i t i o n (%) o f Melanins f r o m S. Sclerotiorum and Ustilago rnaydis .1 Melanin s o u r c e
C
H
N
P
OMe
Ash
S. sclerotiorum
60.3
3.7
1.2 0.9
0.06 0.04
--12.6
0.6 _,2 _,2
62.8
3.4
1.0
HCI h y d r o l . CH=N 2 m e t h y l a t e d
Ustilago maydis CH2N 2 m e t h y l a t e d
12.2
,1 Nicolaus and Piattelli, 1965. *~ I n s u f f i c i e n t sample for ash d e t e r m i n a t i o n .
is virtually unchanged on further HC1 hydrolysis of the pigment, its N content decreasing from 1.2 to 0.9% (table I). Such a low nitrogen content suggests that the origin of the melanin, as proposed b y Jones (1970), is probably catechol rather than DOPA, thus classifying the melanin with those of vegetable origin (Nicolaus and Piattelli, 1965). Indeed, the spectrum of the sclerotinia melanin resembles that of a synthetic pigment prepared b y the enzymic oxidation of a mixture of simple phenols including catechol (Filip et al., 1974) and of catechol alone (Andrews and Pridham, 1967). The similarity is only superficial, however, in that the 1615 cm-' band is weaker than that in the 1200--1300 cm -1 region for the synthetic polymers whereas in melanin the 1615 cm -1 band is stronger (Figs. l b , 2a), and unlike natural melanins, these synthetic polymers are soluble in alkali. The above classification of the sclerotinia melanin as a vegetable pigment is apparently at variance with the findings of Liu et al. (1977) who showed that the pigment from the sclerotia of Sclerotium rolfsii Sacc. contained up to 8% N and yielded indole on alkaline fusion, suggesting that their melanin was of animal origin. However, its large N content and solubility at b o t h high and low pH indicates that it is most probably a melanoprotein (Blois, 1978). The spectra of melanin isolated from acidic media (Figs. l b , 2a) differ considerably from those of the melanin treated with alkali (Figs. lc, 2b). The band at 1700 cm -t disappears, broad absorption at 3300--2400 cm -1 and the band at 1240 cm -~ b e c o m e weaker, and simultaneously a new band appears at 1585 cm -~ and absorption at 1390 cm -~ becomes stronger. These changes are consistent with the conversion of a small proportion of free carboxylic acid groups to carboxylate b y alkali, b u t it is significant that the principal band at 1615 cm -1 is n o t affected b y this treatment, and that considerable intensity still remains at 1240 cm -~ and in the 3300--2400 cm -~ region. To try to identify the origin of these three features, the melanin was repeatedly methylated with diazomethane to give a product with a m e t h o x y l content (12.6%) close to that obtained b y Nicolaus and Piattelli (1965) for methylated melanin from the spores of Ustilago maydis (Table I). The IR spectrum of the methylated sclerotinia melanin (Fig. 2c) shows considerable
212 weakening of b o t h the 1615 cm -1 band and of absorption at 3300--2400 cm -1, a shift of the 1240 cm-' band to 1260 cm-' and the appearance of a new band at 1728 cm -1, spectra run at intermediate stages of methylation confirming that the enhancement at 1728 cm -1 occurred at the expense of the 1615 cm -1 band. The 1260 cm -1 and 1728 cm -~ bands arise from esters, possibly unsaturated or aryl (Bellamy, 1975), leading to the conclusion that the 1615 cm-' band arises from carboxylic acid groups. An alternative assignment of the 1615 cm-' band to carboxylate groups is untenable because of the very small ash content of the pigment (Table I). Ester groups derived from the small number of free carboxylic acid groups absorbing at 1700 cm -~ will also contribute to the 1728 cm-' band. The 1615 cm -1 band lies outside the range of even internally hydrogen-bonded carboxylic acid groups in simple structures (Bellamy, 1975}, but in complex, more highly oxidized systems, such factors as extended conjugation and hydrogen bonding to phenolic OH groups might combine to produce these low frequencies. Certainly, the presence of such groups is strongly indicated b y the loss of absorption between 3500 and 3000 cm -1 in Fig. 2a on salt formation (Fig. 2b), and on methylation (Fig. 2c), and particularly b y the appearance in the spectrum of methylated melanin (Fig. 2c) of new bands in the 1200--1020 cm -1 region which is characteristic for alkylarylether groups. The formation of a methyl ester which led to the assignment of the 1615 cm -~ band to conjugated, strongly hydrogen b o n d e d carboxyl groups invalidates previous assignments of this band in melanin spectra solely to conjugated carbonyl groups (Bonnet and Duncan, 1962) or to chelated quinones (Andrews and Pridham, 1967) because methylation o f such systems would not give rise to a group absorbing at a frequency as high as 1728 cm -1. For the same reason, an assignment to/~-diketones, proposed b y Theng and Posner (1967) for an analogous band at 1610 cm-' in humic acid, is precluded, as is an assignment to graphitic structures suggested b y Friedel and Carlson (1972) for a similar band in coal spectra. The results presented here suggest that the melanin structure might be similar to the oxidized polycyclic catechol p o l y m e r proposed b y Nicolaus and Piattelli (1965) in which phenolic OH and carboxylic acid groups occur. The majority of both groups are mutually involved in strong hydrogen bonds, the remainder being relatively free. It is this latter t y p e which participates in salt formation, a property studied b y Bruenger et al. (1967) and more recently b y Larsson and Tjalve (1978) who concluded that melanin behaves as a weak cation exchanger. In soil, melanin will therefore make a significant contribution to the movement and availability of metal ions in the soil through these readily available carboxyl groups. The strongly hydrogen bonded carboxyl groups are unable to participate in this reaction, b u t the observation that diazomethane can break this b o n d and produce ester and m e t h o x y l groups suggests that these carboxyl groups m a y n o t be permanently unavailable. Indeed, Linhares and Martin (1978), while showing that, in soil, fungal melanins were almost as resistant to biodegradation as humic acids, nevertheless reported a loss of up to 15% of added '4C in 12 weeks, indicating structural decomposition which might
213
alter the hydrogen bond status of the carboxyl groups making them more available in ion exchange reactions. The possible contribution of melanins to the humic fraction o f soil organic matter is currently being actively investigated.
REFERENCES Allen, R.J.L., 1940. The estimation of phosphorus. Biochem. J., 34: 858--865. Andrews, R.S. and Pridham, J.B., 1967. Melanins from DOPA-containing plants. Phytochemistry, 6: 13--18. Belcher, R. and Godbert, A.L., 1954. In: Semi-Micro Quantitative Analysis. Longmans, Green and Co., London, 2rid ed., pp. 155--159. Bellamy, L.J., 1975. The Infrared Spectra of Complex Molecules, 1, Chapman and Hall, London, 3rd ed. Blois, M.S., 1978. The melanins: their synthesis and structure. Photochem. Photobiol. Rev., 3: 115--134. Bonnet, T.G. and Duncan, A., 1962. Infrared spectra of some melanins. Nature, 194: 1078--1079. Bruenger, F.W., Stover, B.J. and Atherton, D.R., 1967. The incorporation of various metal ions into in vivo and in vitro melanin. Radiat. Res., 32: 1--12. Filip, Z., Haider, K., Beutelspacher, H. and Martin, J.P., 1974. Comparisons of IR-spectra from melanins of microscopic soil fungi, humic acids and model phenol polymers. Geoderma, 11: 37--52. Friedel, R.A. and Carlson, G.L., 1972. Difficult carbonaceous materials and their infrared and Raman spectra. Reassignments for coal spectra. Fuel, 51: 194--198. Jones, D., 1970. Ultrastructure and composition of the cell-walls of Sclerotinia sclerotiorum. Trans. Br. Mycol. Soc., 54: 351--360. Kosinkiewicz, B., 1977. Humic-like substances of bacterial origin, I. Some aspects of the formation and nature of humic-like substances produced by Pseudomonas. Acta Microbiol. Pol., 26: 377--386. Larsson, B. and Tjalve, H., 1978. Studies on the melanin affinity of metal ions. Acta Physiol. Scand., 104: 479--484. Linhares, L.F. and Martin, J.P., 1978. Decomposition in soil of the humic acid-type polymers (melanins) of Eurotrium echinulaturn, Aspergillus glaucus sp. and other fungi. Soil Sci. Soc. Am. J., 42:738--743. Liu, T.M.E., Yu, P.H. and Wu, L.C., 1977. Isolation purification and identification of dark pigments in the sclerotia of Sclerotiurn rolfsii Sacc. Chih Wu Pao Hu Hsueh Hui Hui K'an, 19: 223--237. Nicolaus, R.A. and Piattelli, M., 1965. Progress in the chemistry of natural black pigments. Rend. Acad. Sci. Fis. Mat., 32: 83--97. Searcy, R.L., Reardon, J.E. and Foreman, J.A., 1967. A new photometric method for serum urea nitrogen determination. Am. J. Med. Technol., 33: 1---6. Theng, B.K.G. and Posner, A.M., 1967. Nature of the carbonyl groups in soil humic acid. Soil Sci., 104: 191--201.