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Prism conchiolin of modern or fossil molluscan shells. An empire of protein paleization
Comp. Biochem.Physiol., 1968, Vol. 24, pp. 567 to 572. PergamonPress. Printedin GreatBritain
PRISM C O N C H I O L I N OF MODERN OR FOSSIL M O L L U S C A N SHELLS. AN EXAMPLE OF P R O T E I N PALEIZATION* S. B R I C T E U X - G R ~ G O I R E , M. F L O R K I N and CH. GRI~GOIRE Institut L~on Fredericq, Biochimie, Universit~ de Liege (Received 31 ffuly 1967)
Abstract--1. While global nacre conchiolin shows in its composition the predominant concentrations of four amino acids: glycine, alanine, serine and aspartie acid, global prism conehiolin shows a different pattern, characterized by two predominant amino acids: glycine and aspartic acid. 2. Prism conchiolin has been isolated from fossil shells of Pinna a~nis (London Clay, Lower Eocene) and Inoceramus sp. (Gauh, Cretaceous). The comparison of modern and fossil prism conchiolins shows differences in the pattern of amino acid composition. The significance of these differences is discussed. INTRODUCTION THE SOBMICROSCOPICstructure of the conchiolin of the prisms in molluscan shells has been studied in this laboratory by Gr~goire (1960, 1961a, b). The inorganic part of a prism is made up of calcite crystals piled one upon the other, the pile being wrapped into a conchiolin sheath built upon a fibrillar structure covered by a very dense protein component. According to the observations of Wetzel (1900) made on Mytilus and Pinna, the chemical and physical characteristics of prism conchiolin are different to those of nacre conchiolin. According to Roche et al. (1951), prism conchiolin is richer in glycine and tyrosine than nacre conchiolin. Tanaka et al. (1960), in their work on Pinctada martensis, conclude that prism conchiolin contains more phenylalanine and proline, and less alanine than nacre conchiolin. MATERIAL AND METHODS Nacre and prisms have been isolated from the shells of modern Atrina (Pinna) nigra and Pinna nobilis. In order to analyse the conchiolin of the nacre and prisms for their content in proteic amino acids, they were ground in a mortar, washed in boiling water and dried to constant weight at 100°C. The dry powder was decalcified in cold 6 N HC1, added in small portions until all effervescence ceased. The suspension was then dialysed against running tap water and distilled water. After evaporation the content of the dialysis bag was hydrolysed for 24 hr in boiling 6 N HCI. The hydrolysate was evaporated, dissolved in buffer, filtered and submitted to amino acid chromatography on a Beckman Spinco Automatic Analyser. From fossil shells of Pinna a~nis (London Clay, Lower Eocene) and Inoceramus sp. (Gault, Cretaceous) and from modem shells of Atrina nigra and of Pinna nobilis, * A preliminary mention in Florkin (1966a, b). 567
568
S. BRICTEux-GI~GOIRE, M. FLORKIN ANn CH. GR~GOIRE
individual prisms, have been isolated under the binocular. The demineralization of the prisms has been performed in saturated aqueous solutions of Titriplex III and in 2 and 25 parts/100 HC] solutions, leaving transparent, glassy, brittle, sometimes tubular fragments of substance, which are the remains of the prism sheaths. After being washed to remove free amino acids, the isolated sheaths from modern and fossil isolated prisms have been hydrolysed and the products analysed. RESULTS Table I shows the composition of global nacre and prism conchiolins in two modern shells, Atrina (Pinna) nigra and Pinna nobilis. It confirms that global nacre conchiolin shows in its composition the predominant concentrations of four amino acids: glycine, alanine, serine and aspartic acid, as was observed previously in the case of other molluscan shells (Florkin et al., 1961). Global prism conchiolin, on the other hand, shows a different pattern, characterized by two predominant amino acids, glycine and aspartic acid, while alanine and serine represent smaller portions of the total of amino acid residues. The debris of demineralized prisms of fossil shells shows the structure seen in Figs. 1 and 2, recorded photographically under the phase-contrast microscope in aqueous suspensions between slide and cover-glass. In several shreds, the characteristic transverse striation, along which small mineral particles are aligned, is still distinctly recognizable. The results of the analysis of hydrolysed modern and fossil prism conchiolins is shown in Table 2. Fossil prism conchiolin, while showing the high percentage of glycine and aspartic acid characterizing modern prism conchiolin, contains larger proportions of alanine, serine and glutamic acid. Until recently, it had long been accepted that the only organic material remaining in fossils is present in the state approaching complete carbonization. Since free amino acids were first recognized by Abelson in fossils of various ages, it has become apparent that organic biopolymers have been preserved for about 100,000 yr, which was the approximate age of the Pleistocene shell of Mercenaria mercenaria studied by Jones & Vallentyne (1960). They detected in this shell a conchiolin which liberated free amino acids by hydrolysis.
DISCUSSION The data of this paper confirm that animal proteins, identified by the liberation of the component amino acids by hydrolysis, may be preserved in fossils for the very long periods of several geological ages (Florkin et aL, 1961 ; Degens & Love, 1965; Foucart et aL, 1965; Jope, 1967; Voss-Foucart, 1967). As already observed in the comparison of nacre conchiolin of modern and fossil molluscan shells (Florkin et al., 1961), in the comparison of the harder protein layer of modern and fossil Brachiopod shells (Jope, 1967), in the comparison of modern and fossil Gastropod shells (Degens & Love, 1965) and in the comparison of modern and fossil collagens (Wyckoff et al., 1964; Ho, 1965, 1966), the comparison of modern and fossil prism conchiolins shows differences in the pattern of amino acid composition. These differences may of course reflect differences which were already present in the living forms. It appears nevertheless that there are many other possible reasons accounting for the differences. In the course of the chemical
(a)
(b)
FIG. l(a). Atrina (Pinna) nigra Durh. (modern). End-stage of decalcification of a single prism. In the sheath shown here, shrinkage and wrinkling conceal the transverse striation (from Gr6goire, 1961a). Phase contrast × 300. (b) Pinna a~nis (London Clay, Lower Eocene, 60 million years). Ehmgated, folded and wrinkled fragments of tubular prism sheaths. The transverse striation is concealed in part by small mineral fragments resisting demineralization by the chelating agent used, and, as in the modern sample (see Fig. 2(a)) by shrinking of the fragments. Phase contrast x 400.
(a)
(d)
(b)
(c)
FIG. 2(a). Atrina (Pinna) nigra Durh. (modern). Sheath fragments from decalcified prisms showing a transverse striation on rectangular facets (from Gr~goire, 1961a). Phase contrast x 800. (b) and (c) Pinna aff~nis (London Clay, Lower Eocene, 60 million years). Debris of prism sheath facets showing small mineral granules disposed along the transverse striation (see Fig. 1). Phase contrast x 400. (d) Inoceramus sp. (Gault, Cretaceous, 135 million years). Elongated sheath shred left by decalcification of a prism. As in Fig. l(b), Fig. 2(b) and (c), abundant microcrystals, escaping demineralization, subsist, either attached to or embedded in the substance of the sheath. Phase contrast x 400.
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events of the time during which a native protein in a living animal has become a paleoprotein recovered from a fossil (pald~ation, Florkin, 1967), alterations in structure and composition have taken place. It must be pointed out, as mentioned by Gr6goire (1959, 1966), that in many fossil molluscan shells, for instance, conchiolin has been altered in ultrastructure as revealed by the electron microscope. Several kinds of modifications are described by Gr6goire (1966) " . . . in the form of discs, lenticular or spheroidal, pebble-shaped bodies, corpuscles in the form of knobs, perforated membranes". Gr6goire (1964) has exposed fragments of modern Nautilus shells to a few of the factors involved in paleization processes, such as heat and pressure. He has concluded that "heat alone in open air, or associated with pressure produced various stages of ultrastructural degradation of conchiolin similar or identical on the electron microscope scale to those observed consistently in remnants of decalcification of Paleozoic and Mezozoic nautiloid and ammonoid shells". In the field of organic geochemistry, which is concerned with the study of carbonaceous substances in geochemistry, it has only been recognized for a short period that proteic fabrics may be preserved in the structure of a fossil, which allows to set aside the difficulty always present when we try to define the origin of small organic molecules which may be diffusing from outside or have been trapped in the material analysed. In the case of biopolymers found in ritu at their anatomical location, contamination may be due to an invasion of the structure by fossil organisms contemporary to the organism concerned. In fact, such contemporaneous contaminants, intermingled with the conchiolin remnants, have been recorded by electron microscopy. It is therefore important, as has been done in the work on fossil nacre conchiolin and on fossil prism conchiolin referred to above, to rule out, through a thorough control by means of the electron microscope, the possibility of contamination by epizoans, parasites and especially boring predators (Gr6goire, 1966). When we come to the comparison of conchiolins in fossil and modern molluscan shells, we must also keep in mind that when comparing their amino acid composition we are dealing with mixtures of proteins. The pattern referred to above as being characteristic of nacre conchiolin (Florkin et al., 1961) is the pattern of a mixture. The conchiolin concerned can be separated into different components with different amino acid patterns (Gr6goire et al., 1955). A change in the pattern of amino acid composition of a definite proteic fabric defined by its anatomical location in a fossil structure may therefore be either the result of the different degrees of resistance of the different components of the mixture, or of alterations resulting from the removal of end-segments or sidechains of polypeptides in the course of paleization. This may go beyond the stage of denaturation, i.e. of disruption of non-covalent bonds, and involve covalent bond rupture. In such an event, the pattern of the amino acid composition will be altered, but, according to our present knowledge of protein chemistry, it is not to be expected that the primary structure, i.e. the amino acid sequence, the only reliable test of homology (see Florkin, 1966a, b), will be modified. It is therefore imperative to proceed with a study of the paleization of proteins by isolating definite chemical species and studying the changes not only of tertiary structure
572
S. BRICTEUx-GR~GO1RE,M. FLORKINAND CH. GR~GOIRE
but of alterations of the polypeptide chains in order to define the aspects of primary structures which may be compared in homologous protein chains of fossil and m o d e r n organisms. T h e teachings of these data will be of importance for extensions into the past of homologies in the field of proteins. On the other hand, the study of the chemical changes undergone by biopolymers in the transition between native proteins and paleoproteins (paleization) represents an entirely new field of protein chemistry. REFERENCES DEGENS E. T. &; LOVE S. (1965) Comparative studies of amino acids in shell structures of Cryraulus trochiformis Stahl, from the Tertiary of Steinhem, Germany. Nature, Lond. 205, 876-878. FLORKINM. (1966a) Aspects Moldculaires de l'Adaptation et de la Phylogdnie. Masson, Paris. FLORKIN M. (1966b) A Molecular Approach to Phylogeny. Elsevier, Amsterdam. FLORKINM. (1967) Fossil shell "conchiolines" and other preserved biopolymers. In Organic Geochemistry. Methods and Results (Edited by EGLINTONG. ~ MURPHY SISTERMARY T.J.). Springer-Verlag, New York. (In press,) FLORKINM., GR~GOIRECH., BRICTEUX-GP.~COIRES. & SCHOFFENIELSE. (1961) Conchiolines de nacres fossiles. C. r. Acad. Sci., Paris 252, 440 A,A2. , FOUCARTM. F., BRIeTELrx-Gm~GOIRES., JEUNIAUXCH. & FLORKrNM. (1965) Fossil proteins of Graptolites. Life S t . 4, 467-471. GR~GOIRE CH. (1959) A study of the remains of organic components in fossil mother of pearl. Bull. Inst. R. Sci. nat. Belg. 35 (13), 1-14. G~GOIRE CH. (1960) Sur les fourreaux organiques des prismes de Mytilus edulis L. Arch. Int. Physiol. Biochim. 68, 836-837. GR~GOIRE CH. (1961a) Sur la structure submicroscopique de la conchioline associde aux prismes des coquilles de Mollusques. Bull. Inst. Roy. Sci. Nat. Belg. 37, 1-34. GR~GOIRE CH. (1961b) Structure of the conchiolin cases of the prisms in Mytilus edulis Linnd. ~7. Biophys. Biochem. Cytol. 9, 395-400. GR~COIRE CH. (1964) Thermal changes in the Nautilus shell. Nature, Lond. 203, 868-869. GR~OOIRECH. (1966) On organic remains in shells of Paleozoic and Mesozoic Cephalopods (Nautiloids and Ammonoids). Bull. Inst. R. Sci. Nat. Belg. 42, fasc. 39, (3) 1-36. GI~GOIRE CH., DUCH.~TEAUGH. & FLORKINM. (1955) La trame protidique des nacres et des perles. Ann. Ocdanogr. (Paris) 31, 1-36. Ho T. Y. (1965) The amino acid composition of bone and tooth proteins in Late Pleistocene mammals. Proc. nat. Acad. Sci. U.S. 54, 26-31. Ho T. Y. (1966) The isolation and amino acid composition of the bone collagen in Pleistocene mammals. Comp. Biochem. Physiol. 18, 353-358. JONESJ. n . & VALLENTYNEJ. R. (1960) Biochemistry of organic matter--I. Polypeptides and amino acids in fossils and sediments in relation to geothermometry. Geochim. cosmochim. Aeta 21, 1-34. JOPE M. (1967) The protein of Brachiopod shell--II. Shell protein from fossil Articulates: amino acid composition. Comp. Biochem. Physiol. 20, 601-605. ROCHEJ., RANSON G. & EYSERRIC-LAFONM. (1951) Sur la composition des scldroprotdines des coquilles des mollusques (conchiolines). C. r. Soc. Biol. 145, 1474-1477. TANAKA S., HATANOH. & ITASAKAO. (1960) Biochemical studies on pearl--IX. Amino acid composition of conchiolin in pearl and shell. Bull. Chem. Soc..Tapan 33, 543-545. VOss-FoLrCART M. F. (1967) Paldoprotdines des coquilles fossiles de Dinosauriens du Crdtacd supdrieur de Provence. Comp. Biochem. Physiol. (In press.) WETZELG. (1900) Die organische Substanz der Schalen yon Mytilus und Pinna. Z. physiol. Chem. 29, 348-410. WYCKOFF R. W. G., McCAUGHEYW. F. & DOBERENZZ° R. (1964) The amino acid composition of proteins from Pleistocene bones. Biochim. biophys. Acta 93, 374--377.
PRISM C O N C H I O L I N OF MODERN OR FOSSIL M O L L U S C A N SHELLS. AN EXAMPLE OF P R O T E I N PALEIZATION* S. B R I C T E U X - G R ~ G O I R E , M. F L O R K I N and CH. GRI~GOIRE Institut L~on Fredericq, Biochimie, Universit~ de Liege (Received 31 ffuly 1967)
Abstract--1. While global nacre conchiolin shows in its composition the predominant concentrations of four amino acids: glycine, alanine, serine and aspartie acid, global prism conehiolin shows a different pattern, characterized by two predominant amino acids: glycine and aspartic acid. 2. Prism conchiolin has been isolated from fossil shells of Pinna a~nis (London Clay, Lower Eocene) and Inoceramus sp. (Gauh, Cretaceous). The comparison of modern and fossil prism conchiolins shows differences in the pattern of amino acid composition. The significance of these differences is discussed. INTRODUCTION THE SOBMICROSCOPICstructure of the conchiolin of the prisms in molluscan shells has been studied in this laboratory by Gr~goire (1960, 1961a, b). The inorganic part of a prism is made up of calcite crystals piled one upon the other, the pile being wrapped into a conchiolin sheath built upon a fibrillar structure covered by a very dense protein component. According to the observations of Wetzel (1900) made on Mytilus and Pinna, the chemical and physical characteristics of prism conchiolin are different to those of nacre conchiolin. According to Roche et al. (1951), prism conchiolin is richer in glycine and tyrosine than nacre conchiolin. Tanaka et al. (1960), in their work on Pinctada martensis, conclude that prism conchiolin contains more phenylalanine and proline, and less alanine than nacre conchiolin. MATERIAL AND METHODS Nacre and prisms have been isolated from the shells of modern Atrina (Pinna) nigra and Pinna nobilis. In order to analyse the conchiolin of the nacre and prisms for their content in proteic amino acids, they were ground in a mortar, washed in boiling water and dried to constant weight at 100°C. The dry powder was decalcified in cold 6 N HC1, added in small portions until all effervescence ceased. The suspension was then dialysed against running tap water and distilled water. After evaporation the content of the dialysis bag was hydrolysed for 24 hr in boiling 6 N HCI. The hydrolysate was evaporated, dissolved in buffer, filtered and submitted to amino acid chromatography on a Beckman Spinco Automatic Analyser. From fossil shells of Pinna a~nis (London Clay, Lower Eocene) and Inoceramus sp. (Gault, Cretaceous) and from modem shells of Atrina nigra and of Pinna nobilis, * A preliminary mention in Florkin (1966a, b). 567
568
S. BRICTEux-GI~GOIRE, M. FLORKIN ANn CH. GR~GOIRE
individual prisms, have been isolated under the binocular. The demineralization of the prisms has been performed in saturated aqueous solutions of Titriplex III and in 2 and 25 parts/100 HC] solutions, leaving transparent, glassy, brittle, sometimes tubular fragments of substance, which are the remains of the prism sheaths. After being washed to remove free amino acids, the isolated sheaths from modern and fossil isolated prisms have been hydrolysed and the products analysed. RESULTS Table I shows the composition of global nacre and prism conchiolins in two modern shells, Atrina (Pinna) nigra and Pinna nobilis. It confirms that global nacre conchiolin shows in its composition the predominant concentrations of four amino acids: glycine, alanine, serine and aspartic acid, as was observed previously in the case of other molluscan shells (Florkin et al., 1961). Global prism conchiolin, on the other hand, shows a different pattern, characterized by two predominant amino acids, glycine and aspartic acid, while alanine and serine represent smaller portions of the total of amino acid residues. The debris of demineralized prisms of fossil shells shows the structure seen in Figs. 1 and 2, recorded photographically under the phase-contrast microscope in aqueous suspensions between slide and cover-glass. In several shreds, the characteristic transverse striation, along which small mineral particles are aligned, is still distinctly recognizable. The results of the analysis of hydrolysed modern and fossil prism conchiolins is shown in Table 2. Fossil prism conchiolin, while showing the high percentage of glycine and aspartic acid characterizing modern prism conchiolin, contains larger proportions of alanine, serine and glutamic acid. Until recently, it had long been accepted that the only organic material remaining in fossils is present in the state approaching complete carbonization. Since free amino acids were first recognized by Abelson in fossils of various ages, it has become apparent that organic biopolymers have been preserved for about 100,000 yr, which was the approximate age of the Pleistocene shell of Mercenaria mercenaria studied by Jones & Vallentyne (1960). They detected in this shell a conchiolin which liberated free amino acids by hydrolysis.
DISCUSSION The data of this paper confirm that animal proteins, identified by the liberation of the component amino acids by hydrolysis, may be preserved in fossils for the very long periods of several geological ages (Florkin et aL, 1961 ; Degens & Love, 1965; Foucart et aL, 1965; Jope, 1967; Voss-Foucart, 1967). As already observed in the comparison of nacre conchiolin of modern and fossil molluscan shells (Florkin et al., 1961), in the comparison of the harder protein layer of modern and fossil Brachiopod shells (Jope, 1967), in the comparison of modern and fossil Gastropod shells (Degens & Love, 1965) and in the comparison of modern and fossil collagens (Wyckoff et al., 1964; Ho, 1965, 1966), the comparison of modern and fossil prism conchiolins shows differences in the pattern of amino acid composition. These differences may of course reflect differences which were already present in the living forms. It appears nevertheless that there are many other possible reasons accounting for the differences. In the course of the chemical
(a)
(b)
FIG. l(a). Atrina (Pinna) nigra Durh. (modern). End-stage of decalcification of a single prism. In the sheath shown here, shrinkage and wrinkling conceal the transverse striation (from Gr6goire, 1961a). Phase contrast × 300. (b) Pinna a~nis (London Clay, Lower Eocene, 60 million years). Ehmgated, folded and wrinkled fragments of tubular prism sheaths. The transverse striation is concealed in part by small mineral fragments resisting demineralization by the chelating agent used, and, as in the modern sample (see Fig. 2(a)) by shrinking of the fragments. Phase contrast x 400.
(a)
(d)
(b)
(c)
FIG. 2(a). Atrina (Pinna) nigra Durh. (modern). Sheath fragments from decalcified prisms showing a transverse striation on rectangular facets (from Gr~goire, 1961a). Phase contrast x 800. (b) and (c) Pinna aff~nis (London Clay, Lower Eocene, 60 million years). Debris of prism sheath facets showing small mineral granules disposed along the transverse striation (see Fig. 1). Phase contrast x 400. (d) Inoceramus sp. (Gault, Cretaceous, 135 million years). Elongated sheath shred left by decalcification of a prism. As in Fig. l(b), Fig. 2(b) and (c), abundant microcrystals, escaping demineralization, subsist, either attached to or embedded in the substance of the sheath. Phase contrast x 400.
569
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events of the time during which a native protein in a living animal has become a paleoprotein recovered from a fossil (pald~ation, Florkin, 1967), alterations in structure and composition have taken place. It must be pointed out, as mentioned by Gr6goire (1959, 1966), that in many fossil molluscan shells, for instance, conchiolin has been altered in ultrastructure as revealed by the electron microscope. Several kinds of modifications are described by Gr6goire (1966) " . . . in the form of discs, lenticular or spheroidal, pebble-shaped bodies, corpuscles in the form of knobs, perforated membranes". Gr6goire (1964) has exposed fragments of modern Nautilus shells to a few of the factors involved in paleization processes, such as heat and pressure. He has concluded that "heat alone in open air, or associated with pressure produced various stages of ultrastructural degradation of conchiolin similar or identical on the electron microscope scale to those observed consistently in remnants of decalcification of Paleozoic and Mezozoic nautiloid and ammonoid shells". In the field of organic geochemistry, which is concerned with the study of carbonaceous substances in geochemistry, it has only been recognized for a short period that proteic fabrics may be preserved in the structure of a fossil, which allows to set aside the difficulty always present when we try to define the origin of small organic molecules which may be diffusing from outside or have been trapped in the material analysed. In the case of biopolymers found in ritu at their anatomical location, contamination may be due to an invasion of the structure by fossil organisms contemporary to the organism concerned. In fact, such contemporaneous contaminants, intermingled with the conchiolin remnants, have been recorded by electron microscopy. It is therefore important, as has been done in the work on fossil nacre conchiolin and on fossil prism conchiolin referred to above, to rule out, through a thorough control by means of the electron microscope, the possibility of contamination by epizoans, parasites and especially boring predators (Gr6goire, 1966). When we come to the comparison of conchiolins in fossil and modern molluscan shells, we must also keep in mind that when comparing their amino acid composition we are dealing with mixtures of proteins. The pattern referred to above as being characteristic of nacre conchiolin (Florkin et al., 1961) is the pattern of a mixture. The conchiolin concerned can be separated into different components with different amino acid patterns (Gr6goire et al., 1955). A change in the pattern of amino acid composition of a definite proteic fabric defined by its anatomical location in a fossil structure may therefore be either the result of the different degrees of resistance of the different components of the mixture, or of alterations resulting from the removal of end-segments or sidechains of polypeptides in the course of paleization. This may go beyond the stage of denaturation, i.e. of disruption of non-covalent bonds, and involve covalent bond rupture. In such an event, the pattern of the amino acid composition will be altered, but, according to our present knowledge of protein chemistry, it is not to be expected that the primary structure, i.e. the amino acid sequence, the only reliable test of homology (see Florkin, 1966a, b), will be modified. It is therefore imperative to proceed with a study of the paleization of proteins by isolating definite chemical species and studying the changes not only of tertiary structure
572
S. BRICTEUx-GR~GO1RE,M. FLORKINAND CH. GR~GOIRE
but of alterations of the polypeptide chains in order to define the aspects of primary structures which may be compared in homologous protein chains of fossil and m o d e r n organisms. T h e teachings of these data will be of importance for extensions into the past of homologies in the field of proteins. On the other hand, the study of the chemical changes undergone by biopolymers in the transition between native proteins and paleoproteins (paleization) represents an entirely new field of protein chemistry. REFERENCES DEGENS E. T. &; LOVE S. (1965) Comparative studies of amino acids in shell structures of Cryraulus trochiformis Stahl, from the Tertiary of Steinhem, Germany. Nature, Lond. 205, 876-878. FLORKINM. (1966a) Aspects Moldculaires de l'Adaptation et de la Phylogdnie. Masson, Paris. FLORKIN M. (1966b) A Molecular Approach to Phylogeny. Elsevier, Amsterdam. FLORKINM. (1967) Fossil shell "conchiolines" and other preserved biopolymers. In Organic Geochemistry. Methods and Results (Edited by EGLINTONG. ~ MURPHY SISTERMARY T.J.). Springer-Verlag, New York. (In press,) FLORKINM., GR~GOIRECH., BRICTEUX-GP.~COIRES. & SCHOFFENIELSE. (1961) Conchiolines de nacres fossiles. C. r. Acad. Sci., Paris 252, 440 A,A2. , FOUCARTM. F., BRIeTELrx-Gm~GOIRES., JEUNIAUXCH. & FLORKrNM. (1965) Fossil proteins of Graptolites. Life S t . 4, 467-471. GR~GOIRE CH. (1959) A study of the remains of organic components in fossil mother of pearl. Bull. Inst. R. Sci. nat. Belg. 35 (13), 1-14. G~GOIRE CH. (1960) Sur les fourreaux organiques des prismes de Mytilus edulis L. Arch. Int. Physiol. Biochim. 68, 836-837. GR~GOIRE CH. (1961a) Sur la structure submicroscopique de la conchioline associde aux prismes des coquilles de Mollusques. Bull. Inst. Roy. Sci. Nat. Belg. 37, 1-34. GR~GOIRE CH. (1961b) Structure of the conchiolin cases of the prisms in Mytilus edulis Linnd. ~7. Biophys. Biochem. Cytol. 9, 395-400. GR~COIRE CH. (1964) Thermal changes in the Nautilus shell. Nature, Lond. 203, 868-869. GR~OOIRECH. (1966) On organic remains in shells of Paleozoic and Mesozoic Cephalopods (Nautiloids and Ammonoids). Bull. Inst. R. Sci. Nat. Belg. 42, fasc. 39, (3) 1-36. GI~GOIRE CH., DUCH.~TEAUGH. & FLORKINM. (1955) La trame protidique des nacres et des perles. Ann. Ocdanogr. (Paris) 31, 1-36. Ho T. Y. (1965) The amino acid composition of bone and tooth proteins in Late Pleistocene mammals. Proc. nat. Acad. Sci. U.S. 54, 26-31. Ho T. Y. (1966) The isolation and amino acid composition of the bone collagen in Pleistocene mammals. Comp. Biochem. Physiol. 18, 353-358. JONESJ. n . & VALLENTYNEJ. R. (1960) Biochemistry of organic matter--I. Polypeptides and amino acids in fossils and sediments in relation to geothermometry. Geochim. cosmochim. Aeta 21, 1-34. JOPE M. (1967) The protein of Brachiopod shell--II. Shell protein from fossil Articulates: amino acid composition. Comp. Biochem. Physiol. 20, 601-605. ROCHEJ., RANSON G. & EYSERRIC-LAFONM. (1951) Sur la composition des scldroprotdines des coquilles des mollusques (conchiolines). C. r. Soc. Biol. 145, 1474-1477. TANAKA S., HATANOH. & ITASAKAO. (1960) Biochemical studies on pearl--IX. Amino acid composition of conchiolin in pearl and shell. Bull. Chem. Soc..Tapan 33, 543-545. VOss-FoLrCART M. F. (1967) Paldoprotdines des coquilles fossiles de Dinosauriens du Crdtacd supdrieur de Provence. Comp. Biochem. Physiol. (In press.) WETZELG. (1900) Die organische Substanz der Schalen yon Mytilus und Pinna. Z. physiol. Chem. 29, 348-410. WYCKOFF R. W. G., McCAUGHEYW. F. & DOBERENZZ° R. (1964) The amino acid composition of proteins from Pleistocene bones. Biochim. biophys. Acta 93, 374--377.