Fig. 1. Records showing the intensity variations of VHF-radar echoes from height h, observed on 14 March 1977 in Lindau/Harz the characteristic Brunt-Vfiisiil/i period of 8-10 min. The vertical layer displacement was about 300 m and the vertical velocity _+0.6 m s- i Preliminary experiments using a tilted antenna beam and spectrum analyzing range-gated radar signals from 2 km height showed blobs of turbulence cells moving with the horizontal wind velocity o f u p t o 10m s 1. Duringthe 15/16 March 1977, a warm-front approach was followed with the VHF-radar. It is evident that the radar was capable of identifying the front by its downward moving boundary between the warm and cold air already about 12 h in advance of the ground passage of the front. During the operational phase of the SOUSY-VHF-Radar, the sensitivity will be improved by almost 50 dB due to a higher power and larger antenna aperture as well as optimum signal processing [4]
so that reliable echoes from atmospheric heights in excess of 15 km can be observed. From the applicational point of view, even with a low-power VHF-radar (-~ 10kW) and small antenna apertures (-~ 100 m 2) it appears to be practicable to record turbulence and wind profiles continuously from heights of a few hundred meters up to several kilometers. Received August 4, 1977 1. Czechowsky, P., et al. : Proc. 17th Conf. on Radar Meteorology, Amer. Meteor. Soc. 349 (1976) 2. Deft, V.E.: Report of NOAA/ERL Wave Propagation Laboratory, Boulder, August 1972 3. Gossard, E.E.: Radio Sei. 12, 89 (1977) 4. R6ttger, J., Czechowsky, P.: Report MPAE-W-00-77-72 of Max-Planck-Institut ffir Aeronomie, Lindan/Harz (1977)
Two-dimensional NMR Spectroscopy A Powerful Tool for the Investigation of Biopolymers in Solution K. Nagayama and K. Wtithrich Institut fiir Molekularbiologie und Biophysik P. Bachmann and R.R. Ernst Laboratorium ftir Physikalische Chemie, Eidgen~Sssische Technische Hochschule, CH-8093 Ztirich Nuclear magnetic resonance (NMR) has become one of the major methods for the study of biological macromolecules in solution [1]. However, there are many situations where the complexity of the spectra makes their analysis impossible. One reason for the complexity of proton resonance spectra is the severe overlap of spin multiplets. Recently, a new technique has been developed for the efficient separation of multiplets by spreading the spectra in a Naturwissenschaften 64 (1977)
second frequency dimension, leading to two-dimensional (2D) J-resolved 1HNMR spectroscopy [2]. The principal features of 2-D proton spectra can be understood by considering Figure 1. Such a spectrum can be thought to arise in the following manner: The conventional 1-D spectrum, shown as the top trace, is placed along the horizontal line d = 0 in the middle of the 2-D plot. Each multiplet is now rotated by 90 ~ about its
9 by Springer-Verlag 1977
center frequency. This process permits a complete separation of chemical shifts, determining the centers of the multiplet displaced along the horizontal axis, and of multiplet splittings, determining the peak positions along the vertical axis. It is evident that multiplets overlapping in a 1-D spectrum will be separated in a 2-D spectrum whenever they possess different chemical shifts. This renders the analysis of complex spectra rather trivial. Experimentally, this technique requires the perfomaance of a set of N spin-echo experiments of the type 90~ 180~ with k = 0 , 1 , . . . , ( N - 1 ) . Due to refocusing of the processing spin vectors by the 180~ pulse, an echo is generated at time/cz after this pulse [3]. Its decay is sampled at M equidistant time points. The N performed experiments thus produce an N by M data matrix (skz), k = 0 ..... ( N - 1); l= 0,..., ( M - 1 ) . A two-dimensional Fourier transformation of the data matrix produces then a 2-D spectrum which, after some simple conformal mapping, can be brought into the form shown in Figure 1. The secret underlying this technique, which permits separation of the chemical shift and spin-spin coupling effect in two orthogonal frequency dimensions, is the fact that the signal phase at the echo peak is modulated by the multiplet splitting but is independent of the chemical shift as long as the spin coupling is sufficiently weak. Thus, the echo formation is governed by the multiplet splitting alone, whereas the echo decay is influenced by both multiplet splitting and chemical shifts. After separation of chemical shifts and multiplet effects, it is also possible to obtain a completely deconpled protcn spectrum by means of a projection on to the horizontal axis. Here, each proton appears as a single peak representing its chemical shift, as is shown in the bottom traces of Figure 1. Three regions of the spectrum of a D20 solution containing 0.1 M of each of the five amino acids Ala, Ile, Thr, His, and Trp (Fig. 1) are used here to illustrate some features of 2-D J-resolved 1H-NMR at 360MHz, i.e., the aliphatic-proton resonances between 0 and 2 ppm, the crowded region from 2.7 to 3.3 ppm shown in Figure l b, and the aromatic resonances between 6.8 and 7.7 ppm. Four methyl resonances can be recognized between 0 and 2 ppm. These are a triplet at 0.84 ppm and a doublet at 0.88 ppm which come from Ile, a doublet of Yhr 581
/
/
/
7
/
6
/
5
4
a
/
/
/
3
2
1
/
~ [ppm]
/
/ 7~ / b
/
/
/
/
/
/
3.3
3.2
3.1
3.0
2,~}
2,8
/
~ [ppm]
Fig. 1.a) Two-dimensional J-resolved 360-MHz ~H-NMR spectrum of a D20 solution containing 0.1 M Ala, Ile, Thr, His, and Trp, pH 10.5, T=25 ~ An absolute-value spectrum is shown. The spectral resolution is improved in both frequency directions by digital filtering. In this representation, the individual components of a given mnltiplet are located on a line perpendicular to the chemical shift axis. Projection of the spectrum along these lines produces a "homonuclear broad band decoupled" 1-D ~H-NMR spectrum, as shown in the bottom trace. For comparison, the conventional 1-D spectrum is shown in the top trace, b) Expanded representation of the spectral region from 2.7 to 3.4ppm at 1.16ppm and a doublet of Ala at 1.29 ppm. While the two highest-field multiplets are partially overlapped in the 1-D spectrum, all four methyl resonances are fully resolved both in the 2-D spectrum and in the homonuclear decoupled spectrum. In addition, there are three one-proton multiplets corresponding to the ?methylene protons and the/7-methine proton of Ile at 1.12, 1.37, and 1.73 ppm. These resonances appear as broad multi582
plets in the 1-D spectrum and as sharp peaks in the homonuclear decoupled spectrum. With the vertical scale used in Figure 1a they cannot readily be seen in the 2-D spectrum. Between 2.7 and 3.3 ppm (Fig. 1 b), there are two doublets of doublets corresponding to the/7-methylene protons of His at 2.78 and 2.93 ppm. The [?-methylene protons of Trp give rise to similar four-line resonances at 3.02 and 3.20 ppm. The dou-
Nets at 3. I0 and 3.26 ppm correspond to the s-protons of Ile and Thr, respectively. All the multiplets are readily recognized in the 2-D spectrum. The ]7-methylene resonances of His and Trp still contain some partially resolved long-range spinspin couplings of < 1 Hz. The spectral resolution in the homonuclear decoupled spectrum is greatly improved compared with the conventional 1-D spectrum. The aromatic region contains the doublets of the indole-ring protons H(4) and H(7) of Trp at 7.66 and 7.44 ppm, the singlets of the imidazole-ring protons H(2) and H(4) of His at 7.62 and 6.68 ppm, the H(2) singlet resonance of Trp at 7.15 ppm and the H(6) and H(5) triplet resonances of Trp at 7.18 and 7.10 ppm. While they are in part overlapped in the conventional 1-D spectrum, the five resonances of Trp are well resolved both in the 2-D spectrum and the homonuclear decoupled spectrum. On the other hand, with the horizontal scale of Figure 1 a, the His resonances are not readily seen in the 2-D spectrum and appear as very weak lines in the homonuclear decoupled representation. The distorted signal intensities in 2-D and in projected 1-D spectra are caused by a combination of several effects which are all dependent on the resonance line width. These effects are presently under investigation. The additional resonances in Figure 1 a are the quartet of the Ala s-proton at 3.47 ppm, and the rather broad, in the 1-D spectrum only partially resolved multiplets of the c~-protons of His and Trp and of the /7-proton of Thr at 3.50, 363, and 3.93ppm, respectively. The singlet of HDO is at 4.73 ppm. 2-D J-resolved spectroscopy is particularly suited for the spread of high-field proton spectra as it depends on the weak-coupling assumption. This technique is only one example of a broad and very promising class of 2-D experiments with numerous applications in almost all branches of NMR. Some other knowr/ applications cover the 2-D resolution of 13C spectra in liquids and solids [4], the elucidation : of energy-level schemes by 2-D-correlated spectroscopy [5, 6], the indirect high-sensitivity detection of resonance [7] and the measurement of forbidden multiple quantum transitions [6, 8]. It is expected that at least some of these techniques will soon become routine tools in N M R spectroscopy. Research grants of the ETH and the Swiss
Naturwissenschaften 64 (1977)
9 by Springer-Verlag 1977
National Science Foundation are gratefully acknowledged.
Received July 25, 1977 1. Wfithricb, K.: NMR in Biological Research: Peptides and Proteins. Amsterdam: North-Holland 1976; Dwek, R.A. : Nuclear Magnetic Resonance in Biochemistry. Oxford: Clarendon Press 1973 2. Aue, W.P., Karhan, J., Ernst, R.R.: J. Chem. Phys. 64, 4226 (1976); Kumar, A., et al.: Proc. XIXth Congres Amp6re, Heidelberg, p. 473 (1976) 3. Hahn, E.L., Maxwell, D.E. : Phys. Rev. 88, 1070 (1952)
4. Mfiller, L., Kumar, A., Ernst, R.R.: J. Chem. Phys. 63, 5490 (1975); Bodenhausen, G., Freeman, R., Turner, D.L.: ibid. 65, 839 (1976); Miiller, L., Kumar, A., Ernst, R.R.: J. Magn. Resonance 25, 383 (1977); Hester, R.K., etal.: Phys. Rev. Lett. 36, 1081 (1976) 5. Jeener, J.: Amp+re Int. Summer School II, Basko Polje (1971) 6. Aue, W.P., Bartholdi, E., Ernst, R.R.: J. Chem. Phys. 64, 2229 (1976) 7. Maudsley, A.A., Ernst, R.R. : Chem. Phys. Lett. (in press) 8. Vega, S., Shattuck, T.W., Pines, A. : Phys. Roy. Lett. 37, 43 (1976) ; Wokaun, A., Ernst, R.R. : submitted
Calcium-binding Peptide in Dinosaur Egg Shells G. Krampitz, K. Weise~, A. Potz, J. Engels, T. Samata, K. Becker, and M. Hedding Abteilung ftir Biochemie, Institut fiir Anatomie, Physiologic und Hygiene der Haustiere, Universitfit Bonn, D-5300 Bonn G. Flajs Institut ftir PalS,ontologie der Universit/it, D-5300 Bonn Ca-binding macromolecules have been found in the organic matrix of biomineralizates, e.g., egg shells [1] and mollusk shells [2, 3], as well as in coccoliths of recent species ['4]. De Jong et al. [5] also described a Ca-binding polysaccharide extracted from the coccoliths of subfossil Emiliania huxleyi. The Ca-binding properties of the soluble fractlonagree with the hypothesis that this material plays a role in cellular regulation of calcium carbonate crystallization. Ca-binding macromolecules might act as nucleators of CaCO3 precipitation [5]. We report on a still actively Cabinding peptide preserved in egg shells of the Maastrichtian-stage late-Cretaceous dinosaurs. We extracted and fractionated water-soluble substances from egg shells of Hypselosaurus priscus Matheron collected near Rcusset (Aix-en-Provence, France). The procedures applied were reported recently [6]. Ca-binding material was charged with *SCaZ+ and located on cellulose plates (TLC) by autoradiography. A ninhydrinpositive zone carrying 45Ca was purified to homogeneity in more than 10 solvent systems (TLC). In contrast to corresponding comPonents in egg shells of recent species, e.g., Gallus domesticus [7], the Cabinding molecule did not contain phosphate, sulfate, sugars, sugar derivatives, or hydrocarbons9 Extraction of the shells with chloroform-methanol yielded, however, 9
t
Naturwissenschaften 64 (1977)
green and yellow pigments separable by TLC. Extracts of soil and stone where the shell fragments were found did not contain any Ca-binding substances. Upon HC1 hydrolysis of the Ca-binding substance, primarily amino acids were recovered. The main constituent of the Cabinding peptide was glycine (> 90%). Minor components were histidine, lysine, and unknown ninhydrin-positive substances eluted near the positions of tyrosine, histidine, and arginine on an automatic aminoacid analyzer (Beckman Multichrom B). The unknown ninhydrin-positive components might be artifacts resulting from diagenesis. The apparent molecular size of the purified Ca-binding peptide by gel filtration was ca. 1500-2300 daltons as determined on Bio-Gel P-6 with peptides (~-
MSH, apamine) as references. End-group determinations revealed glycine to be in both the C- and N-terminal positions. The Ca-binding peptide in dinosaur egg shells resembled the comparable material from recent species in various respects (e.g., amino acid sequence and Ca 2+-binding capacity). Comparable observations were made by Hausmanns [8] while studying the composition of enzymatically prepared fragments of Ca-binding systems L'om egg shells, uterine fluid, and uterine mucosal cells of hens. The relatively low molecular size of the fossil peptide could be due to degradation, possibly by diagenetical processes. The particularly large proportion of glycine might reflect the ingress of an organic molecule serving as a mineral-ion carrier and might also be associated with the active acquisition of mineral ions in calcification processes [9]. The close association of this peptide with CaCO3 might have contributed to the (partial) preservation of this molecule over a period of about 70 million years. Received August 30, 1977 i. Krampitz, G., Engels, J., in: Proteins and Related Subjects, Vol. 22, p. 327 (ed. H9 Peeters). London: Pergamon Press 1975 2. Crenshaw, M.A. : Biomineralization 6~ 6 (1972) 3. Krampitz, G., et al., in: The Mechanisms of Mineralization in the Invertebrates arrd Plants, p. 155 (eds. N. Watanabe and K.I~: Wilbur). Univ. of South Carolina Press 1976 4. De Jong, L.W., et al. : ibid., p. 135 5. De Jong, L.W., et al. : Eur. J. Biociaem. 70, 611 (1976) 6. Krampitz, G., Engels, J. : Biomineralization 8, 21 (1975) 7. Krampitz, G., et al. : Arch. Geflfigelkde: (in press) 8. Hausmanns, G. : Dissertation Bonn 1977 9. Robinson, C., et al. : Calc. Tiss. Res. 23, 19 (1977); Urry, D.W.: Proc. Nat. Acad. Sci. USA 68, 810 (1971)
Raman-Spektrum des P~--Ions in Alkalijodidschmelzen W. Bues, M. Somer, W. Brockner und D. Grtinewald Anorganisch-Chemisches Institut der Technischen Universitfit, D-3392 Clausthal-Zellerfeld In den Polyphosphiden Sr3Pa4 und Ba3P14, deren Darstellung und Struktur von Dahlmann und v. Schnering [1, 2] publiziert wurde, liegen isolierte P~--Gruppierungen mit C3v-Symmetrie vor. Von den gleichen Autoren [2, 3] wurden auch
9 by Springer-Verlag 1977
die IR- und Raman-Feststoffspektren gemessen und auf die ~_hnlichkeit des Ba3P14-IR-Spektrums mit dem des P4S3Molekiils [4], das analog dem P73--Ion aufgebaut ist, hingewiesen. Raman-Spektren yon Gardner [5] zeigen, dag P4S3 als Mole583
so that reliable echoes from atmospheric heights in excess of 15 km can be observed. From the applicational point of view, even with a low-power VHF-radar (-~ 10kW) and small antenna apertures (-~ 100 m 2) it appears to be practicable to record turbulence and wind profiles continuously from heights of a few hundred meters up to several kilometers. Received August 4, 1977 1. Czechowsky, P., et al. : Proc. 17th Conf. on Radar Meteorology, Amer. Meteor. Soc. 349 (1976) 2. Deft, V.E.: Report of NOAA/ERL Wave Propagation Laboratory, Boulder, August 1972 3. Gossard, E.E.: Radio Sei. 12, 89 (1977) 4. R6ttger, J., Czechowsky, P.: Report MPAE-W-00-77-72 of Max-Planck-Institut ffir Aeronomie, Lindan/Harz (1977)
Two-dimensional NMR Spectroscopy A Powerful Tool for the Investigation of Biopolymers in Solution K. Nagayama and K. Wtithrich Institut fiir Molekularbiologie und Biophysik P. Bachmann and R.R. Ernst Laboratorium ftir Physikalische Chemie, Eidgen~Sssische Technische Hochschule, CH-8093 Ztirich Nuclear magnetic resonance (NMR) has become one of the major methods for the study of biological macromolecules in solution [1]. However, there are many situations where the complexity of the spectra makes their analysis impossible. One reason for the complexity of proton resonance spectra is the severe overlap of spin multiplets. Recently, a new technique has been developed for the efficient separation of multiplets by spreading the spectra in a Naturwissenschaften 64 (1977)
second frequency dimension, leading to two-dimensional (2D) J-resolved 1HNMR spectroscopy [2]. The principal features of 2-D proton spectra can be understood by considering Figure 1. Such a spectrum can be thought to arise in the following manner: The conventional 1-D spectrum, shown as the top trace, is placed along the horizontal line d = 0 in the middle of the 2-D plot. Each multiplet is now rotated by 90 ~ about its
9 by Springer-Verlag 1977
center frequency. This process permits a complete separation of chemical shifts, determining the centers of the multiplet displaced along the horizontal axis, and of multiplet splittings, determining the peak positions along the vertical axis. It is evident that multiplets overlapping in a 1-D spectrum will be separated in a 2-D spectrum whenever they possess different chemical shifts. This renders the analysis of complex spectra rather trivial. Experimentally, this technique requires the perfomaance of a set of N spin-echo experiments of the type 90~ 180~ with k = 0 , 1 , . . . , ( N - 1 ) . Due to refocusing of the processing spin vectors by the 180~ pulse, an echo is generated at time/cz after this pulse [3]. Its decay is sampled at M equidistant time points. The N performed experiments thus produce an N by M data matrix (skz), k = 0 ..... ( N - 1); l= 0,..., ( M - 1 ) . A two-dimensional Fourier transformation of the data matrix produces then a 2-D spectrum which, after some simple conformal mapping, can be brought into the form shown in Figure 1. The secret underlying this technique, which permits separation of the chemical shift and spin-spin coupling effect in two orthogonal frequency dimensions, is the fact that the signal phase at the echo peak is modulated by the multiplet splitting but is independent of the chemical shift as long as the spin coupling is sufficiently weak. Thus, the echo formation is governed by the multiplet splitting alone, whereas the echo decay is influenced by both multiplet splitting and chemical shifts. After separation of chemical shifts and multiplet effects, it is also possible to obtain a completely deconpled protcn spectrum by means of a projection on to the horizontal axis. Here, each proton appears as a single peak representing its chemical shift, as is shown in the bottom traces of Figure 1. Three regions of the spectrum of a D20 solution containing 0.1 M of each of the five amino acids Ala, Ile, Thr, His, and Trp (Fig. 1) are used here to illustrate some features of 2-D J-resolved 1H-NMR at 360MHz, i.e., the aliphatic-proton resonances between 0 and 2 ppm, the crowded region from 2.7 to 3.3 ppm shown in Figure l b, and the aromatic resonances between 6.8 and 7.7 ppm. Four methyl resonances can be recognized between 0 and 2 ppm. These are a triplet at 0.84 ppm and a doublet at 0.88 ppm which come from Ile, a doublet of Yhr 581
/
/
/
7
/
6
/
5
4
a
/
/
/
3
2
1
/
~ [ppm]
/
/ 7~ / b
/
/
/
/
/
/
3.3
3.2
3.1
3.0
2,~}
2,8
/
~ [ppm]
Fig. 1.a) Two-dimensional J-resolved 360-MHz ~H-NMR spectrum of a D20 solution containing 0.1 M Ala, Ile, Thr, His, and Trp, pH 10.5, T=25 ~ An absolute-value spectrum is shown. The spectral resolution is improved in both frequency directions by digital filtering. In this representation, the individual components of a given mnltiplet are located on a line perpendicular to the chemical shift axis. Projection of the spectrum along these lines produces a "homonuclear broad band decoupled" 1-D ~H-NMR spectrum, as shown in the bottom trace. For comparison, the conventional 1-D spectrum is shown in the top trace, b) Expanded representation of the spectral region from 2.7 to 3.4ppm at 1.16ppm and a doublet of Ala at 1.29 ppm. While the two highest-field multiplets are partially overlapped in the 1-D spectrum, all four methyl resonances are fully resolved both in the 2-D spectrum and in the homonuclear decoupled spectrum. In addition, there are three one-proton multiplets corresponding to the ?methylene protons and the/7-methine proton of Ile at 1.12, 1.37, and 1.73 ppm. These resonances appear as broad multi582
plets in the 1-D spectrum and as sharp peaks in the homonuclear decoupled spectrum. With the vertical scale used in Figure 1a they cannot readily be seen in the 2-D spectrum. Between 2.7 and 3.3 ppm (Fig. 1 b), there are two doublets of doublets corresponding to the/7-methylene protons of His at 2.78 and 2.93 ppm. The [?-methylene protons of Trp give rise to similar four-line resonances at 3.02 and 3.20 ppm. The dou-
Nets at 3. I0 and 3.26 ppm correspond to the s-protons of Ile and Thr, respectively. All the multiplets are readily recognized in the 2-D spectrum. The ]7-methylene resonances of His and Trp still contain some partially resolved long-range spinspin couplings of < 1 Hz. The spectral resolution in the homonuclear decoupled spectrum is greatly improved compared with the conventional 1-D spectrum. The aromatic region contains the doublets of the indole-ring protons H(4) and H(7) of Trp at 7.66 and 7.44 ppm, the singlets of the imidazole-ring protons H(2) and H(4) of His at 7.62 and 6.68 ppm, the H(2) singlet resonance of Trp at 7.15 ppm and the H(6) and H(5) triplet resonances of Trp at 7.18 and 7.10 ppm. While they are in part overlapped in the conventional 1-D spectrum, the five resonances of Trp are well resolved both in the 2-D spectrum and the homonuclear decoupled spectrum. On the other hand, with the horizontal scale of Figure 1 a, the His resonances are not readily seen in the 2-D spectrum and appear as very weak lines in the homonuclear decoupled representation. The distorted signal intensities in 2-D and in projected 1-D spectra are caused by a combination of several effects which are all dependent on the resonance line width. These effects are presently under investigation. The additional resonances in Figure 1 a are the quartet of the Ala s-proton at 3.47 ppm, and the rather broad, in the 1-D spectrum only partially resolved multiplets of the c~-protons of His and Trp and of the /7-proton of Thr at 3.50, 363, and 3.93ppm, respectively. The singlet of HDO is at 4.73 ppm. 2-D J-resolved spectroscopy is particularly suited for the spread of high-field proton spectra as it depends on the weak-coupling assumption. This technique is only one example of a broad and very promising class of 2-D experiments with numerous applications in almost all branches of NMR. Some other knowr/ applications cover the 2-D resolution of 13C spectra in liquids and solids [4], the elucidation : of energy-level schemes by 2-D-correlated spectroscopy [5, 6], the indirect high-sensitivity detection of resonance [7] and the measurement of forbidden multiple quantum transitions [6, 8]. It is expected that at least some of these techniques will soon become routine tools in N M R spectroscopy. Research grants of the ETH and the Swiss
Naturwissenschaften 64 (1977)
9 by Springer-Verlag 1977
National Science Foundation are gratefully acknowledged.
Received July 25, 1977 1. Wfithricb, K.: NMR in Biological Research: Peptides and Proteins. Amsterdam: North-Holland 1976; Dwek, R.A. : Nuclear Magnetic Resonance in Biochemistry. Oxford: Clarendon Press 1973 2. Aue, W.P., Karhan, J., Ernst, R.R.: J. Chem. Phys. 64, 4226 (1976); Kumar, A., et al.: Proc. XIXth Congres Amp6re, Heidelberg, p. 473 (1976) 3. Hahn, E.L., Maxwell, D.E. : Phys. Rev. 88, 1070 (1952)
4. Mfiller, L., Kumar, A., Ernst, R.R.: J. Chem. Phys. 63, 5490 (1975); Bodenhausen, G., Freeman, R., Turner, D.L.: ibid. 65, 839 (1976); Miiller, L., Kumar, A., Ernst, R.R.: J. Magn. Resonance 25, 383 (1977); Hester, R.K., etal.: Phys. Rev. Lett. 36, 1081 (1976) 5. Jeener, J.: Amp+re Int. Summer School II, Basko Polje (1971) 6. Aue, W.P., Bartholdi, E., Ernst, R.R.: J. Chem. Phys. 64, 2229 (1976) 7. Maudsley, A.A., Ernst, R.R. : Chem. Phys. Lett. (in press) 8. Vega, S., Shattuck, T.W., Pines, A. : Phys. Roy. Lett. 37, 43 (1976) ; Wokaun, A., Ernst, R.R. : submitted
Calcium-binding Peptide in Dinosaur Egg Shells G. Krampitz, K. Weise~, A. Potz, J. Engels, T. Samata, K. Becker, and M. Hedding Abteilung ftir Biochemie, Institut fiir Anatomie, Physiologic und Hygiene der Haustiere, Universitfit Bonn, D-5300 Bonn G. Flajs Institut ftir PalS,ontologie der Universit/it, D-5300 Bonn Ca-binding macromolecules have been found in the organic matrix of biomineralizates, e.g., egg shells [1] and mollusk shells [2, 3], as well as in coccoliths of recent species ['4]. De Jong et al. [5] also described a Ca-binding polysaccharide extracted from the coccoliths of subfossil Emiliania huxleyi. The Ca-binding properties of the soluble fractlonagree with the hypothesis that this material plays a role in cellular regulation of calcium carbonate crystallization. Ca-binding macromolecules might act as nucleators of CaCO3 precipitation [5]. We report on a still actively Cabinding peptide preserved in egg shells of the Maastrichtian-stage late-Cretaceous dinosaurs. We extracted and fractionated water-soluble substances from egg shells of Hypselosaurus priscus Matheron collected near Rcusset (Aix-en-Provence, France). The procedures applied were reported recently [6]. Ca-binding material was charged with *SCaZ+ and located on cellulose plates (TLC) by autoradiography. A ninhydrinpositive zone carrying 45Ca was purified to homogeneity in more than 10 solvent systems (TLC). In contrast to corresponding comPonents in egg shells of recent species, e.g., Gallus domesticus [7], the Cabinding molecule did not contain phosphate, sulfate, sugars, sugar derivatives, or hydrocarbons9 Extraction of the shells with chloroform-methanol yielded, however, 9
t
Naturwissenschaften 64 (1977)
green and yellow pigments separable by TLC. Extracts of soil and stone where the shell fragments were found did not contain any Ca-binding substances. Upon HC1 hydrolysis of the Ca-binding substance, primarily amino acids were recovered. The main constituent of the Cabinding peptide was glycine (> 90%). Minor components were histidine, lysine, and unknown ninhydrin-positive substances eluted near the positions of tyrosine, histidine, and arginine on an automatic aminoacid analyzer (Beckman Multichrom B). The unknown ninhydrin-positive components might be artifacts resulting from diagenesis. The apparent molecular size of the purified Ca-binding peptide by gel filtration was ca. 1500-2300 daltons as determined on Bio-Gel P-6 with peptides (~-
MSH, apamine) as references. End-group determinations revealed glycine to be in both the C- and N-terminal positions. The Ca-binding peptide in dinosaur egg shells resembled the comparable material from recent species in various respects (e.g., amino acid sequence and Ca 2+-binding capacity). Comparable observations were made by Hausmanns [8] while studying the composition of enzymatically prepared fragments of Ca-binding systems L'om egg shells, uterine fluid, and uterine mucosal cells of hens. The relatively low molecular size of the fossil peptide could be due to degradation, possibly by diagenetical processes. The particularly large proportion of glycine might reflect the ingress of an organic molecule serving as a mineral-ion carrier and might also be associated with the active acquisition of mineral ions in calcification processes [9]. The close association of this peptide with CaCO3 might have contributed to the (partial) preservation of this molecule over a period of about 70 million years. Received August 30, 1977 i. Krampitz, G., Engels, J., in: Proteins and Related Subjects, Vol. 22, p. 327 (ed. H9 Peeters). London: Pergamon Press 1975 2. Crenshaw, M.A. : Biomineralization 6~ 6 (1972) 3. Krampitz, G., et al., in: The Mechanisms of Mineralization in the Invertebrates arrd Plants, p. 155 (eds. N. Watanabe and K.I~: Wilbur). Univ. of South Carolina Press 1976 4. De Jong, L.W., et al. : ibid., p. 135 5. De Jong, L.W., et al. : Eur. J. Biociaem. 70, 611 (1976) 6. Krampitz, G., Engels, J. : Biomineralization 8, 21 (1975) 7. Krampitz, G., et al. : Arch. Geflfigelkde: (in press) 8. Hausmanns, G. : Dissertation Bonn 1977 9. Robinson, C., et al. : Calc. Tiss. Res. 23, 19 (1977); Urry, D.W.: Proc. Nat. Acad. Sci. USA 68, 810 (1971)
Raman-Spektrum des P~--Ions in Alkalijodidschmelzen W. Bues, M. Somer, W. Brockner und D. Grtinewald Anorganisch-Chemisches Institut der Technischen Universitfit, D-3392 Clausthal-Zellerfeld In den Polyphosphiden Sr3Pa4 und Ba3P14, deren Darstellung und Struktur von Dahlmann und v. Schnering [1, 2] publiziert wurde, liegen isolierte P~--Gruppierungen mit C3v-Symmetrie vor. Von den gleichen Autoren [2, 3] wurden auch
9 by Springer-Verlag 1977
die IR- und Raman-Feststoffspektren gemessen und auf die ~_hnlichkeit des Ba3P14-IR-Spektrums mit dem des P4S3Molekiils [4], das analog dem P73--Ion aufgebaut ist, hingewiesen. Raman-Spektren yon Gardner [5] zeigen, dag P4S3 als Mole583
