NOTIZEN 5 8 5 Conformation Analysis of the Polypeptides
in the Thylakoid Membrane
E I K E H . P E T Z E L a n d W I L H E L M M E N K E
Max-Planck-Institut für Züchtungsforschung (Erwin-Baur- Institut), Köln-Vogelsang
(Z. Naturforsch. 27 b, 585—586 [1972] ; received February 10, 1972)
The ultraviolet circular dichroism spectrum of a chloroplast fraction of Antirrhinum ma jus has recently been published The fraction was obtained from stroma-freed chloroplasts by ultrasonic treatment and fractioning centrifugation2-3. The spectrum shows extrema of ellipticity at 194 ( + ), 208 ( - ) and 222
( —) nm. Thus it is similar to a protein with a con- siderable a-helix content. G R E E N F I E L D and F A S M A N 4
have calculated circular dichroism spectra for proteins of different conformations by linear superposition of reference spectra. These authors used poly-L-lysine in a, ß and random coil conformations for reference. A comparison with the spectra of G R E E N F I E L D and F A S -
MAN 4 shows that the spectrum of the fragments of the thylakoid membrane closely resembles that of a pro- tein with 42 percent a-helix, 40 percent random coil and 18 percent ^-structure (Fig. 1). The biggest dif-
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Fig. 1. Comparison of the experimental (• • •) with a calcu- lated ( ) spectrum obtained by linear superposition of polylysine reference spectra according to GREENFIELD and FASMAN. The 121 points which mark the experimental curve are arithmetic means of 5 — 10 registrations from 5 prepara-
tions.
ference between the experimental and calculated spec- tra consists in a red shift of the positive extremum from 191 to 194 nm. In order to exclude experimental errors in this comparison, we have measured the spectra of poly-L-lysine in its three conformations with the same apparatus 1 in which we measured the spectra of the thylakoid fragments. The preparative methods have been described previously 2> 3.
A comparable red shift, besides other distortions, has been described several times for optical rotation dispersion and circular dichroism spectra of mem-
Requests for reprints should be sent to Prof. Dr. W. MENKE, MPI für Züchtungsforschung, D-5000 Köln 30.
branes. This red shift has recently been attributed to light scattering at the membranes5-12. However, the approximate average particle size in the suspensions used in this investigation is only 100 Ä. Consequently, light scattering should not be the main cause of the observed red shift.
An attempt to more accurately determine the con- formation parameters, by means of a linear-least-squa- res-fit, did not yield a better fit to the experimental spectrum, because its characteristic shape was not maintained in the calculated spectrum (Fig. 2). How-
ever, a closer fit is obtained if one allows a shift of the reference spectra on the wavelength-scale (Fig. 3).
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Fig. 2. Approximation of the experimental spectrum by means of a linear-least-squares-fit. The conformation parameters Pi of the theoretical function y (A) = Pa • Ra (2) + Pß-Rß (A) + Py-Ry(2) are varied to give the minimum sum of error squa- res. Ra, R/3, Ry are the reference spectra for a-helix, ß- structure and random coil. The fit was obtained by computer.
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Fig. 3. Approximation of the experimental spectrum by means of a non-linear-least-squares-fit of the chloroplast circular dichroism spectrum. The theoretical function is: y (A) = Pa - Ra ( A - A a) + P y - R yU - A y ) . Ai are the shift parameters. A j < 0 means a shift to longer, A j > 0 means
a shift to shorter wavelengths.
With a shift of the a-helix spectrum by 2 nm to longer wavelengths and of the random coil and /^-structure spectra by 1 respectively 2 nm to shorter wavelengths, a content of 35 percent a-helix, 24 percent random coil and 27 percent /^-structure are observed. However, this result is also unsatisfactory, as the conformation parameters do not yield 100 percent. Furthermore, 27
5 8 6 NOTIZEN percent /J-structure and a 35 percent a-helix content should show up in the IR-spectrum.
As scattering effects are probably not the cause for the red shift, an unknown conformation or influences
1 W . M E N K E , Z . N a t u r f o r s c h . 2 5 b . 8 4 9 [ 1 9 7 0 ] .
2 C. G . K A N N A N G A R A , D . V A N W Y K, a n d W . M E N K E , Z . N a -
turforsch. 25b. 613 [1970].
3 A . R A D U N Z , G . H . S C H M I D, and W . M E N K E , Z . N a t u r -
forsch. 26 b, 435 [1971].
4 N . GREENFIELD and G . D . FASMAN, B i o c h e m i s t r y 8 . 4 1 0 8 [ 1 9 6 9 ] ,
5 J. M . STEIN and S. FLEISCHER, P r o c . nat. A c a d . Sei. U S A 5 8 . 1 2 9 2 [ 1 9 6 7 ] .
0 D. W. URRY and T. H. Ji, Arch. Biochem. Biophysics 128,
8 0 2 [ 1 9 6 8 ] .
7 D . W . U R R Y and J. K R I V A C I C, P r o c . nat. A c a d . Sei. U S A
65.845 [1970].
which are due to intermolecular interactions must be considered. The eigendichroism of the lipids adds only very little to the circular dichroism in the far UV and can thus be disregarded.
8 A . S . SCHNEIDER, M. - J . T . SCHNEIDER, and K . ROSENHECK.
Proc. nat. Acad. Sei. USA 66, 793 [1970].
9 D . W . U R R Y, L . M A S O T T I, and J. K R I V A C I C, B i o c h e m . Bio-
phys. Res. Comm. 41, 521 [1970] ; Biochim. biophysica Acta [Amsterdam] 241, 600 [1971].
10 J. C. REINERT and J. L. DAVIS, Biochim. biophysica Acta [Amsterdam] 241,921 [1971],
1 1 M . GLASER and S . J. SINGER, B i o c h e m i s t r y 1 0 , 1 7 8 0
[1971].
1 2 D . J. G O R D O N and G . H O L Z W A R T H, P r o c . nat. A c a d . Sei.
USA 68, 2365 [1971].
p-coumaroyl-ttieso-tartaric acid from Spinach Chloroplast Preparations
W A L T E R O E T T M E I E R a n d A D E L H E I D H E U P E L
Abteilung für Biologie, Ruhr-Universität Bochum
(Z. Naturforsch. 27 b, 586—587 [1972] ; received February 1972)
In a recent publication1 we reported on the iso- lation and identification of flavonoids and cinnamic acid derivatives from spinach chloroplast preparations.
These cinnamic acid derivatives and flavonoids are obtained as low molecular fractions I and II from a water extract — called S^eth — from ether treated lyophilized chloroplasts, which is assumed to contain functional components of the photosynthetic electron transport chain between photosystem I and ferre- doxin 2'3. We have discussed the possibility that a phenolic compound might be the prosthetic group of this functional component (possibly the primary ac- ceptor) and also the relation of our phenolic com- pounds to other fractions of various activity isolated from chloroplasts by other authors 1.
We have shown that the chromophoric group of low molecular fraction I consists of two esters of p-couma- ric acid 1. The chemical nature of the compounds esteri- fied to p-coumaric acid remained to be elucidated. We wish to report here on their identification.
The p-coumaric acid derivatives of low molecular fraction I, absorbing at 312 nm, were obtained from the water extract of ether treated lyophilized chloro- plasts as previously described After alkaline hydro- lysis of its chromophore, the resultant alkaline solu- tion was passed through a Dowex 50 WX 8 column and the p-coumaric acid extracted with ether1. The compound, which was originally esterified to p-couma- ric acid, could now be found in the water phase. It was identified as meso-tartaric acid by paper chro-
Requests for reprints should be sent to Dr. W. OETTMEIER, Ruhr-Universität Bochum, Abteilung für Biologie, D-4630 Bochum-Querenburg, Postfach 2148.
matography (Paper Macherey und Nagel MN 2261 FF:
n-butanol — formic acid —water 4 : 1 : 5 (by volume, upper phase) Rf = 0.12 and n-butanol — 2% boric acid —ethyl acetate — formic acid 15 : 5 : 5 : 2 (by vo- lume) Rf = 0.10. 7?/-values of DL-tartaric acid in these solvent systems are 0.19 and 0.15, respectively.
Spots were detected by spraying with a 0.04% solution of bromophenol blue in 95% ethanol, pH 5).
The identity of the chromophoric group of low mole- cular fraction I with p-coumaroyl-meso-tartaric acid
COOH I H - C - O H
H — C — O — CO — CH = CH — ^ > - O H COOH
was proved by comparison with a synthetic sample.
Synthetic p-coumaroyl-meso-tartaric acid was obtained according to the procedure of T A D E R A et al.4. Its spectral and chromatographic data are identical with those we had recently reported on the chromophoric group of low molecular fraction I, for example Amax (methanol) 312 nm, after addition of sodium methylate 358 nm, i?/-values in thin layer chromatography on cel- lulose in solvent systems n-butanol — acetic acid —wa- ter 4 : 1 : 5 (upper phase), water and n-butanol — 2 N NH3 1 : 1 (upper phase)
p-coumaroyl-iweso-tartaric acid and its acetyl deriva- tive have been very recently isolated by T A D E R A et al. 4' 5 from spinach leaves.
From the data Wu et al. 6 published for the chromo- phoric group ("P-compound") of a fluorescent protein from spinach chloroplasts, which stimulates photophos- phorylation, it seems quite likely that "P-compound"' is identical with or related to p-coumaroyl-meso-tar- taric acid.
We are indebted to Prof. Dr. A. TREBST and the "Deutsche Forschungsgemeinschaft" for supporting this work.
NOTIZEN 5 8 7
1 W . O E T T M E I E R a n d A . HEUPEL, Z . N a t u r f o r s c h . 2 7 b , 1 7 7 [1972].
2 G . R E G I T Z , R . B E R Z B O R N, a n d A . TREBST, P l a n t a B e r l i n 9 1 , 8 [1970].
3 G . R E G I T Z a n d W . OETTMEIER, P r o g r e s s in P h o t o s y n t h e s i s Research, Stresa 1971, in press.
4 K . T A D E R A , Y . S U Z U K I , F . K A W A I, a n d H . M I T S U D A, A g r . Biol. Chem. 34, 517 [1970].
3 K . T A D E R A a n d H . M I T S U D A, A g r . B i o l . C h e m . 3 5 , 1 4 3 1 [1971].
6 M . W u , J. M Y E R S a n d H . S . F O R R E S T, A r c h . B i o c h e m . B i o - physics 140, 391 [1970].
Zur Thermodynamik der Konformations- änderungen des Hämoglobins Thermodynamics of the Conformational Changes
of Hemoglobine
K . S . T R I N T S C H E R
Institut für Biophysik der Akademie der Wissenschaften der UdSSR
(Z. Naturforsch. 27 b, 587—588 [1972] ; eingeg. am 24. Dezember 1971)
Die Prozesse der Oxy- und Desoxygenation werden von Struktur-energetischen Veränderungen begleitet.
Die Oxygenation geht mit Volumsverlust ( — AV) und Wärmeabgabe ( — AQ) vor sich, während die Desoxy- genation von Volumsvergrößerung (+ AV) und Wärmeaufnahme (+ AQ) begleitet wird. Beschreiben wir diese Prozesse in Form von Gleichungen, die außer den üblichen diemischen Symbolen energetische und geometrische Symbole enthalten:
Hb + 02- J ( ? - z l F = Hb0,. ( l a ) Hb02 + zl(? + /IF = Hb + 02. ( l b ) Die Gin. (1 a) und (1 b) stellen drei parallel vor sich gehende Prozesse dar:
Glei- Chemische Energie- Volumen- chung Reaktion umwandlung veränderung
(1 a) Hb + 02 t / H b - A 0 VEb-AV
= H b 02 = u Hb02 = ^Hb02
(1 b) HbOo £ / H b 02+ ^ ( ? VRb02 + AV
— Hb + 02 = U Hb = VKb
uHb, FHb, ^Hb02> Vnb02 bedeuten innere Energien und Volumina der Hb- und Hb02-Moleküle. Bestim- men wir nun den Unterschied zwischen den inneren Energien von Hb und Hb02 durch die Strukturenergie des Hb-Moleküls:
UHb — C^Hb02 — AUStr.Hb • ( 2 )
Der Übergang von Wärme in Strukturenergie wird durch die Strukturarbeit des Hämoglobin-Moleküls ge- leistet :
AQ = ^ S t r . H b = AUstr, H b - (3)
Die Strukturarbeit stellt Volumsarbeit dar, entspre- chend der Gleichung:
Astr,Eb = N0-pEb-AV. (4)
Hier bedeuten N0 die A v o g a d r o - Konstante, pHb den intramolekularen Druck des Hämoglobin-Moleküls.
Da nun die Strukturarbeit der absorbierten Wärme äquivalent ist und diese der Reaktionswärme des Oxy- genationsprozesses gleich ist, so folgt:
A Q
Nehmen wir für die Hb- und Hb02-Moleküle Kugel- form an mit den Radien 1:
riib = 25 Ä und rHb02 Sä 22 Ä,
so erhalten wir für die den Molekülen äquivalenten Kugeln die folgenden Volumswerte:
FHb ^ y /-Hb -71 = 6-10-2 0cm3,
F//bo2 S3 I 'Hbo* -n = 4,3 • 1 0 -2W . (6) und hieraus
AV = FHÖ — FHb02 = 1/7' 1 0- 2 0 cm3. (7) Die Reaktionswärme bei der Kombination eines Mols Sauerstoff mit einem Mol Hämoglobin ist gleich 2- 3:
AQ ^ 1,4 • 10—4 cal. Mol- 1. (8) Setzen wir nun die Zahlenwerte für AV, AQ, N0 und
den Umwandlungsfaktor: 1 cal ^ 41 cm3• atm, in Gl.
(5) ein, so erhalten wir:
1,4-104-41 , s
PHb = 6 • 1023 • 1,7 • 10"20 = 5 7 a t m- (9) Wie aus Gl. (9) folgt, ist der intramolekulare Druck des Hämoglobin-Moleküls sehr gering; er ist etwa um zwei Größenordnungen kleiner als der innere Druck leicht assoziierender Flüssigkeiten, der in den Grenzen von 3-103 —6 103 atm liegt. Hieraus läßt sich der Schluß ziehen: der intramolekulare Druck des Hämoglobin- Moleküls bezieht sich auf den Druck zwischen den be- weglichen Monomeren, den zwei a- und zwei y5-Ketten des Hämoglobin-Moleküls. Tatsächlich sind Konforma- tionsänderungen nur am intakten Hb-Molekül festzu- stellen, während die einzelnen a- und /3-Ketten keine Volumsänderung bei der Oxy- bzw. Desoxygenation er- fahren. Dieses Ergebnis gestattet es von einer „Ultra- Mobilität" der a- und /^-Ketten im Hb-Molekül zu spre- chen, und offenbar ist es diese Ultra-Mobilität, die der unmittelbaren Umwandlung der absorbierten Wärme in Volumsarbeit zugrunde liegt.
Da das Hb-Molekül kein Energiespender ist, so kann der mit der Sauerstoffbindung verknüpfte Energie- anteil nur durch das 02-Molekül in Form der kineti- schen Energie geliefert werden:
Mn • v2 3
2 = j N0k T^ 6-102 cal. Mol'1.
5 8 8 NOTIZEN Dieser Energieanteil ist im Vergleich zu AQ sehr ge- ring und kann daher vernachlässigt werden. Die vom Hämoglobin-Molekül absorbierte Wärme wandelt sich über die Strukturarbeit in Strukturenergie um, wäh- rend die Strukturenergie bei Volumsschrumpfung des Hämoglobin-Moleküls unmittelbar in Wärme übergeht.
Beide Prozesse werden durch die folgenden Gleichun- gen wiedergegeben:
Hb + 02 - zf t/str.Hb ~N0-p0-AV = Hb02 + AQ. (10 a)
Hb02 + N0• pub•AV=Eb + 02 + AU&tr>nb . (10b) Hier bedeuten AQ und 02 die Wärme- und Sauer- stolf-Abgabe, p0 den Nullwert des intramolekularen Drucks, nachdem das Hämoglobin-Molekül sein Maxi- mal-Volumen erreicht hat, und pHb = 57 atm, den Ma- ximalwert des intramolekularen Drucks zu Beginn der Volumsarbeit des Hämoglobinmoleküls.
1 M. F. PERUTZ, Structure of Hemoglobin. In "Protein 2 F. J. W. ROUGHTON et al., Bioch. J. 30, 2117 [1936].
Structure and Function". N3, Brockhaven Symposia in Bio- 3 G. ANTONINI, Physiol. Reviews, Vol. 45, N 1, 123 [1965].
logy, Upton N.Y., 1960.
