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A study of the yellow astaxanthin-proteins of lobster carapace
Comp. Biochem. Phy.siol. Vol. 71B, pp. 243 Printed in Great Britain
to
247, 1982
0305-0491/82/020243-05503.00/0 Pergamon Press Ltd
A STUDY OF THE YELLOW A S T A X A N T H I N - P R O T E I N S O F LOBSTER CARAPACE P. F. ZAGALSKY Biochemistry Department, Bedford College, Regent's Park, London NW1 4NS, U.K. (Received 25 June 1981)
Abstract--1. Astaxanthin-proteins absorbing in the 400 nm region, larger in size than yellow pigment (2m,x 409 nm), are present in extracts of lobster carapace. The subunits of the pigments are of the same size as those of the blue pigment, ~-crustacyanin. 2. The subunit composition of yellow pigment (2..... 409 nm), is studied. The pigment is polydisperse in electrophoresis and is composed of two different types of subunit, one type of which is common to ~-crustacyanin.
INTRODUCTION
In addition to the crustacyanins absorbing in the 600 n m region (Jencks & B u t e n , 1964; Cheesman et al., 1966; K u h n and Kiihn, 1967), a yellow pigment with an absorption m a x i m u m at 409 nm has been characterised from lobster carapace (Jencks & Buten, 1964; Buchwald & Jencks, 1968). The hysochromically shifted absorption spectrum of this pigment results from exciton coupling between the 20 or so astaxanthin prosthetic groups, stacked like a pile of plates (Buchwald & Jencks, 1968; Salares et al., 1977). The pigment has been located, by extraction and in resonance R a m a n studies, at the outer surface of the carapace (Salares et al., 1977). Although distinct in amino acid composition, it shows immunological similarities with the crystacyanins (Buchwald & Jencks, 1968); the subunit composition has not been published. Photoacoustic spectroscopy of lobster shell has provided evidence for yellow carotenoproteins b o t h at the outer surface (Zmax 425 rim) and at the interior (2m,x 390 rim) of the endocuticle ( M a c k e n t h u n et al., 1979). A carotenoprotein corresponding to the 390 nm pigment has not been reported in shell extracts. The present paper reports a study of yellow pigments of the carapace of the lobster, H o m a r u s gammarus (L). MATERIALS AND METHODS Lobster Homarus 9ammarus (L) pigments, extracted as described by Quarmby et al., 1977, were dialysed against 0.25 M phosphate buffer (KHzPOg-Na2HPO4), pH6.9, and passed through a column of DEAE-cellulose (DE32, Whatman, England) equilibrated with the same buffer. The column was washed thoroughly with the 0.25 M buffer to remove ~-, fl- and 7-crustacyanins. The remaining pigment, green-yellow in colour, was eluted with 1 M K.C1 in 0.05 M phosphate buffer, pH 6.9 (Jencks & Buten, 1968), and fractionated on columns of Sephadex G200 and Agarose 4B (Pharmacia, Upsala) equilibrated with the latter solution, performed as in Zagalsky & Herring (1977). These and subsequent operations were carried out in the cold and dark. Polyacrylamide gel electrophoresis (PAGE), in the presence and absence of 0.17~, dodecylsulphate (SDS) or 6 M 243
urea, was carried out in rods or slabs using the method of Laemmli & Favre (1973). The composition of gels are expressed as follows (Hjert6n, 1962): T denotes the total weight of monomer (acrylamide plus N,N'-methylenebisacrylamide) per 100 ml of solution and C denotes the amount of N,N'-methylenebisacrylamide expressed as a percentate (w/w) of the total amount of monomer. Gradient-gel slabs ( T = 7.9 22.6, C = 2.9) were made with spacer gels (T = 3.9, C = 2.9) both with and without 0.1'~'J~,SDS; there were variations in the linearity of gradients between slab batches. Isoelectric focusing (IEF) was performed in rods ( T = 5, C = 3.0) with ampholine pH 3 10 (L.K.B.), as described in the Pharmacia booklet, Polyacrylamide gel electrophoresis, laboratory techniques; The Coomassie brilliant blue R250 staining and destaining procedure given in the booklet was followed. The pigments, partially purified by gel filtration, were studied as follows: 1. Pigments were examined in PAGE both in gradient gel slabs run for 2000 vhr and in rods (T = 8.4, C = 2.9). Pigmented bands of interest (including ~-crustacyanin) were excised, sliced longitudinally and incubated (a) for 1.5 hr at 37°C in 27~, SDS-0.125 M tris HCI, pH 6.8, - 5 % sucrosebromophenol blue (BPB)(10/~g/ml), with and without 5°~ mercaptoethanol (ME), (b) for 1.0hr at 37'C in 6 M urea 0.125 M tris-HCl, pH 6.8, - B F B (10/lg/ml). The slices were then placed both longitudinally and vertically on the spacer gel of the appropriate SDS-acrylamide gradient or 6 M urea slab gel (T = 8.4, C = 2.9), covered with the incubation solution and electrophoresis performed at 100V. A molecular weight calibration kit (M.W. 14.4 x 103-94 × 103; Pharmacia) was treated as in (a), above, and run on a separate gel slab. Gels made with and without SDS were stained as in Bordier & Crettol-Jarvinen (1979), and those containing urea by the method of Cleveland et al. (1977). 2. Yellow protein ()'max 409 nm) was focused as a fairly broad band near the bottom (surface pH 4.6-4.8) of IEF rods. The bands were excised, incubated with 0.068 M tris HC1, pH 6.8-5~o sucrose-BFB (10 tlg/ml) for 45 min at 20°C. The pieces were then placed onto the spacer gel ol rods (T = 8.4, C = 2.9), covered with incubation solution and PAGE performed with the discontinuous buffer system at 30 V overnight. The yellow band was excised, sliced longitudinally and treated as for slices in (1), above. 3. Chromatofocusing of yellow protein (2,,ax 409 rim), partly purified by Sephadex G200, on polybuffer exchanger PBE T M 94 was carried out as described in the Pharmacia chromatofocusing kit booklet, with polybuffer 74 as eluant.
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Fig. l. Gel filtration elution profile of lobster pigments on Sephadex G200. The sample was the pigment eluting in DEAE cellulose chromatography with 1 M KCI 0.05 M phosphate buffer, pH 6.9 (see Methods section), following passage of 0.25 M phosphate buffer, pH 6.9. The column (75 x 3.2cm) was equilibrated with the eluant, l M KCI 0.05 M phosphate buffer, pH 6.9, in the dark at 3C. Sample volume: 3 ml. Fraction volume: 3 ml. Flow rate: 10 ml/hr. Curves represent the extinctions of fractions at 400 nm, , and at 630 nm,- . . . . . ; the values of the ratio of extinctions at 400 and 280nm (E40o/280) are given, . Inset: rechromatography of pooled fractions B, yellow pigment (2 ...... 409 nm).
Following passage of the polybuffer the strongly adsorbed pigment was partly eluted with 0.5 M KCI. The pigment was concentrated by ultrafiltration, dialysed against 0.068 M tris HC1, pH 6.8, and subjected to discontinuous PAGE in rods, or alternatively, gradient gel slabs for 2000vhr. Pigment bands of interest were excised and treated as for slices described in (1), above.
0.8 60
It is clear from the gel filtration studies (Figs. 1 3) that, in addition to yellow protein (2 ...... 409nm), extracts of lobster carapace contain other pigments of larger molecular size absorbing in the 400 nm region. Pigments eluting prior to the yellow protein on columns of Sephadex G200 (Fig. 1) were partially resolved on columns of Agarose 4B (Fig. 2) and subsequent r e c h r o m a t o g r a p h y on columns of Sephadex G200 (Fig. 3) into the following fractions: a minor red pigment, excluded from Agarose 4B, with 2 ...... at 398 4- 1 nm, 470 _+ 2 nm and 620 4- 5 nm of relative extinction 1.0/0.8/0.35; a fraction (Pigment Yl) larger in size than ;¢-crustacyanin, of Stokes radius 130 A, with ,:,...... at 398 4- 1 n m and 470 4- nm of relative extinction 1.0/0.7; a fraction (Pigment Y j smaller in size than =-crustacyanin, of Stokes radius 60/L with )....... at 401 _+ 1 nm and an inflexion at 470 nm. Pigments Y1 and Y2 were incompletely resolved from residual ~-crustacyanin and from each other. In gradient gel electrophoresis Pigment Yt moved as a red yellow band and Pigment Y2 as a yellow zone of lower and greater mobility, respectively, than ~-crustacyanin (Fig. 4). Both pigments gave predominantly two subunits in S D S - P A G E (Fig. 5), identical in size to those of =-crustacyanin; the proteins did not dissociate in 6 M urea.
120
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Fig. 2. Gel filtration elution profile of pigment fraction A (see Fig. 1) on Agarose 4B. The column (75 x 3.2 cm) was equilibrated and run as stated in Fig. 1. Curves represent the extinction of fractions at 400 nm, , and at 630 nm, . . . . . . . Values of E40o,,280 are given,
The best preparations of yellow protein (2 ...... 409 nm) obtained in gel filtration had a ratio of absorptions at 409 and 280 n m (E409/280) of 3.4. The partly purified protein eluted in a symmetrical but b r o a d band (Fig. 1 inset) with a Stokes of ca. 37,&. The behaviour of the protein in gel electrophoresis is unusual. In PAGE, both with continuous (tris borate-EDTA)* and discontinuous buffer systems, the pigment showed considerable spread and uneven protein staining within the pigment region (Fig. 4). This
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Fig. 3. Gel filtration elution profiles of pigment fractions C and D (see Fig. 2) on Sephadex G200. (A) Pigment fraction D. (B) Pigment fraction C. The column (75 x 3.2 cm) was equilibrated and run as stated in Fig. I. Curves represent the extinction of fraction at 400 nm, - , and at 630 nm, . . . . . . . Values of 54-oo128o are given, . Pigment fractions Y~ and Y2 are referred to in the text.
A study of the yellow astaxanthin-proteins of lobster carapace
245
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Fig. 4. Electrophoresis of yellow protein (2,,ax 409 nm) following gel filtration on Sephadex G200 and of pigment fraction A (Fig. 1). Yellow protein (2max 409 nm): (a) PAGE (T = 8.4, C = 2.9). (b) PAGE (T = 10.9, C = 2.9). (c) IEF, pH 3-10. (d) PAGE (T = 8.4, C = 2.9) of excised pigment zone following IEF. (e) PAGE in gradient gel for 2000 vhr. The zone of yellow protein is indicated between the arrows. Pigment fraction A (Fig. 1): (f) PAGE in gradient gel for 2000vhr. YI, pigment Y1; Y2, pigment Y2; ct,~-crustacyanin.
occurred also in IEF at pH 3-10, in PAGE following IEF, and in gradient gel electrophoresis run for 2000vhr to concentrate the protein at its exclusion point. Two groups subunits were evident when the gel slice of yellow protein obtained in PAGE or IEF or gradient PAGE was subjected to S D ~ P A G E in an orientation perpendicular to the direction of mobility in the former gels (slice horiz. Fig. 5). Subunits (Type 1) of an identical size to those of ct-crustacyanin (1.9 x 104 and 2.2 x 104) were present fairly evenly across the slice region and, in some gels, a component of greater mobility, of apparent molecular weight 1.3 x 104. In addition, slanting protein bands (usually two), of highest molecular size for the yellow pigment of lowest mobility in the first gel, covered the pigmented zone of the slice. These bands were resolved into 4 components (Type II subunits) of apparent molecular size 5 x 104-8 x 104, not always present in the same relative proportions, when the electrophoresis was performed with the slice in an orientation parallel to that of the first run (Slice vert. Fig. 51. The electrophoresis pattern was unaltered when slices were treated with mercaptoethanol. In 6 M urea-PAGE, slices run in a perpendicular orientation to that in the first direction (PAGE, IEF or gradient PAGE) similarly showed slanting protein bands, together with the subunits common to ct-crustacyanin. The latter on occasions appeared only as minor components (Fig. 5). The protein was eluted from polybuffer PBE TM 94 with an altered absorption spectrum. The fraction, with 2 .... at 411 nm and a minor inflexion at 470 nm, showed a stronger absorption at 280 nm (with a value
of E411/280 of 1.5) than the starting sample. It gave both Type I and Type II subunits in SDS-PAGE (Fig. 5). DISCUSSION
Recent interest in the distribution of astaxanthinproteins within lobster carapace has focused attention to their possible photobiological function (Salares et al., 1977; Mackenthun et al., 1979). It has been proposed that the yellow protein at the outermost layer of the endocuticle acts as a light receptor passing energy inwards to pigments of bathochromically shifted spectra (Salares et al., 1977; Mackenthun et al., 1979). Photoacoustic spectroscopy (PAS) of carapace reveals an anisotropic distribution of pigments with a steady shift in absorption maximum from 450 to 625 nm, from outer to inner parts of the endocuticle, together with pigments hypsochromically shifted in spectra at the outer (~'max 425 nm) and inner surface (2ma x 390nm). Infrared resonance Raman spectroscopy has provided evidence that pigments with absorption maxima at even longer wavelength than ct-crustacyanin may be present in the carapace (Nelson & Carey, 1981). It is uncertain whether additional pigments to those already identified (ct- fl- and ),-crustacyanins and yellow protein (~'max 409 nm)) are yet to be isolated or whether interactions of the latter proteins within the carapace can account for the above observations. The absorption spectra of pigment fractions Yz and Yz isolated in the present study cover the lower wavelength absorption within the endocuticle apparent in PAS. It would be facile to conclude that pigment Y1
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Fig. 5. Electrophoresis of yellow protein (2 .... 409 nm) in gels containing SDS or 6 M urea. (aF(n) SDS- PAGE in gradient gels. (aF(c) excised pigment zone following PAGE ( T = 8.4, C = 2.9) (Fig. 4a): (a) slice horiz., (b) slice horiz., treated ME, (c) slice vert. Apparent molecular sizes of Group II subunits (see text) derived from a plot of RBVB VS log molecular weight: (1) 7.9 x 10 4 (2) 6.6 x 104 (3) 5.5 x 104 (4) 4.9 x 104. (d) Yellow protein purified by chromatofocusing and PAGE in gradient gel for 2000 vhr: excised pigment zone, slice vert. (eF(fl pigment Ya (e) and YI (f) excised from gel, Fig. 4(f). (g) e-crustacyanin excised from PAGE gel; subunit nomenclature (C2, C~, A,, As and A3) as in Quarmby et al. (1977). (h) Following IEF, pigment zone excised (Fig. 4c) and subjected to PAGE [T = 8.4, C = 2.9). Pigment zone excised from PAGE gel (Fig. 4d), slice vert. (i) As in (e). (j) As in (h), slice horiz. (k) Yellow protein purified by chromatofocusing and PAGE (T = 8.4, C = 2.9). Pigment zone excised from PAGE gel, slice vert. (1) As in (h), slice horiz., treated ME. (m) Pigment zone following PAGE in gradient gel for 2000 vhr {Fig. 4el, slice horiz. (n) As in (e). (oh(t) 6 M urea PAGE (T = 8.4, C = 2.9). (o) As in (m). (p) As in (e). (q) As (m), slice vert. (r) As in (j). (s) As in (h). (t) As in (e).
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A study of the yellow astaxanthin-proteins of lobster carapace
247
A heterogeneous mixture of yellow proteins, is located at the outer part of the endocuticle and 7 × 104-10 × l04 in molecular size, is consistent with pigment Y2 at the inside. It is not possible to decide, the molecular weight studies (Buchwald & Jencks, however, whether either pigment is native to the cara1968) of this fraction. pace or is formed during the extraction procedure, since crustacyanin denatures to pigments of similar size and spectrum at high pH (Jencks & Buten, 1964; Buchwald & Jencks, 1968), a condition which may REFERENCES arise during extraction. The unstable yellow protein (2 .... 409 nm) has a BORDIER C. & CRETTOL-JARVINENA. (1979) Peptide mapping of heterogeneous protein samples, d. biol. Chem. molecular weight of ca. 90,000 and a minimum mol25,1, 2565-2567. ecular weight, based on the astaxanthin content, of BUCHWALD M. & JENCKS W. P. (1968) Properties of the 4400 (Buchwald & Jencks, 1968). The protein comcrustacyanins and the yellow lobster shell pigment. Bioponent of the pigment thus represents a weight of chemistry 7, 844-859. about 77,000, assuming 20 astaxanthin prosthetic CHEESMAND. F., ZAGALSKVP. F. & CECCALDIH. J. (1966) groups. The amino acid composition is distinct from Purification and properties of crustacyanin. Proc. R. Soc. :t-crustacyanin (Buchwald & Jencks, 1968) and from BI64, 130-151. the individual subunits of this protein (Quarmby et CLEVELAND D. W., FISHER S. G., KIRSCHNER M. W. & al., 1977). The heterogeneous nature of this yellow LAEMMLI U.K. (1977) Peptide mapping by limited proprotein component, regardless of the method used to teolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. biol. Chem. 252, 1102 1106. separate the pigment, is evident in the present study. HJERTEN S. (1962) Molecular sieve chromatography on Two types of subunit, based on molecular size, may polyacrylamide gels, prepared according to a simplified be distinguished. The subunits of Type II, of apparent procedure. Archs. Biochem. Biophys. Suppl. I, 147-t51. size 50 × 103 80 × 103, must be responsible for the JENCI,:SW. P. & BUTENB. 0964) The denaturation of crushigh glutamate content (Buchwald & Jencks, 1968) of tacyanin. Archs. Biochem. Biophys. 107, 511 520. the pigment. The proteins of Type I, common to KUHN R. & KOHN H. (1967) Quat~irstruktur und farbe yon ~-crustacyanin, account for the immunological crosscrustacyanin. Eur. J. Biochem. 2, 349-360. reactions between the yellow protein and the crustaLAEMMLIU. K. & FAVREM. (1973) Maturation of the head of the bacteriophage T,,. I. DNA packaging events. J. cyanins (Buchwald & Jencks, 1968). molec. Biol. 80, 575-599. The polydisperse behaviour of the pigment in the MACKENTHUN M. t., TOM R. D. & MOORE T. A. (1979) electrophoretic investigations may be explained if the Lobster shell carotenoprotein organisation in situ quaternary structure is variable and consists of pairs explored by photoacoustic spectroscopy. Nature, Lond. of subunits, each pair containing a peptide from each 278, 861-862. of the two types of subunit. The relative proportions NELSONW. H. & CAREVP. R. (1971) Infrared excited resoof subunits within each class in any given preparation nance Raman Spectra of lobster shell pigments in situ. d. would then depend on the particular mixture of pigRaman Spectrosc. In press. ment molecules present. It would seem reasonable to QUARMBYR., NORDEND. A., ZAGALSKYP. F., CECCALDIH. suppose that the number of astaxanthin in carotenoid J. & DAUMASR. (1977) Studies on the quaternary structure of the lobster exoskeleton carotenoprotein, crustamicelles of pigment molecules of different subunit cyanin. Comp. Biochem. Physiol. 56B, 55-61. composition may also be variable (about the mean of 20), unless the smaller group II subunit (5 × 10 4) is SALARESV. R., YOUNGN. M., BERNSTEINH. J. & CAREYP. R. (1977) Resonance Raman spectra of lobster shell caroable to associate with the same size of carotenoid tenoproteins and a model astaxanthin aggregate. A possmicelle as the larger (8 × 104 ) subunit; it would ible photobiological function for the yellow protein. Bioappear from the electrophoretic results that the subchemistry 16, 4751-4756. units of lower molecular size (Type I) are more readily ZAGALSKVP. F. & HERRINGP. J. (1977) Studies of the blue lost from the association during purification proastaxanthin-proteins of Velella velella (Coelenterata: cedures and may therefore not play so important a Chondrophora). Philos. Trans. R. Soc. London. Ser. B. 279, 289-326. role in stabilisation of the carotenoid micelle.
to
247, 1982
0305-0491/82/020243-05503.00/0 Pergamon Press Ltd
A STUDY OF THE YELLOW A S T A X A N T H I N - P R O T E I N S O F LOBSTER CARAPACE P. F. ZAGALSKY Biochemistry Department, Bedford College, Regent's Park, London NW1 4NS, U.K. (Received 25 June 1981)
Abstract--1. Astaxanthin-proteins absorbing in the 400 nm region, larger in size than yellow pigment (2m,x 409 nm), are present in extracts of lobster carapace. The subunits of the pigments are of the same size as those of the blue pigment, ~-crustacyanin. 2. The subunit composition of yellow pigment (2..... 409 nm), is studied. The pigment is polydisperse in electrophoresis and is composed of two different types of subunit, one type of which is common to ~-crustacyanin.
INTRODUCTION
In addition to the crustacyanins absorbing in the 600 n m region (Jencks & B u t e n , 1964; Cheesman et al., 1966; K u h n and Kiihn, 1967), a yellow pigment with an absorption m a x i m u m at 409 nm has been characterised from lobster carapace (Jencks & Buten, 1964; Buchwald & Jencks, 1968). The hysochromically shifted absorption spectrum of this pigment results from exciton coupling between the 20 or so astaxanthin prosthetic groups, stacked like a pile of plates (Buchwald & Jencks, 1968; Salares et al., 1977). The pigment has been located, by extraction and in resonance R a m a n studies, at the outer surface of the carapace (Salares et al., 1977). Although distinct in amino acid composition, it shows immunological similarities with the crystacyanins (Buchwald & Jencks, 1968); the subunit composition has not been published. Photoacoustic spectroscopy of lobster shell has provided evidence for yellow carotenoproteins b o t h at the outer surface (Zmax 425 rim) and at the interior (2m,x 390 rim) of the endocuticle ( M a c k e n t h u n et al., 1979). A carotenoprotein corresponding to the 390 nm pigment has not been reported in shell extracts. The present paper reports a study of yellow pigments of the carapace of the lobster, H o m a r u s gammarus (L). MATERIALS AND METHODS Lobster Homarus 9ammarus (L) pigments, extracted as described by Quarmby et al., 1977, were dialysed against 0.25 M phosphate buffer (KHzPOg-Na2HPO4), pH6.9, and passed through a column of DEAE-cellulose (DE32, Whatman, England) equilibrated with the same buffer. The column was washed thoroughly with the 0.25 M buffer to remove ~-, fl- and 7-crustacyanins. The remaining pigment, green-yellow in colour, was eluted with 1 M K.C1 in 0.05 M phosphate buffer, pH 6.9 (Jencks & Buten, 1968), and fractionated on columns of Sephadex G200 and Agarose 4B (Pharmacia, Upsala) equilibrated with the latter solution, performed as in Zagalsky & Herring (1977). These and subsequent operations were carried out in the cold and dark. Polyacrylamide gel electrophoresis (PAGE), in the presence and absence of 0.17~, dodecylsulphate (SDS) or 6 M 243
urea, was carried out in rods or slabs using the method of Laemmli & Favre (1973). The composition of gels are expressed as follows (Hjert6n, 1962): T denotes the total weight of monomer (acrylamide plus N,N'-methylenebisacrylamide) per 100 ml of solution and C denotes the amount of N,N'-methylenebisacrylamide expressed as a percentate (w/w) of the total amount of monomer. Gradient-gel slabs ( T = 7.9 22.6, C = 2.9) were made with spacer gels (T = 3.9, C = 2.9) both with and without 0.1'~'J~,SDS; there were variations in the linearity of gradients between slab batches. Isoelectric focusing (IEF) was performed in rods ( T = 5, C = 3.0) with ampholine pH 3 10 (L.K.B.), as described in the Pharmacia booklet, Polyacrylamide gel electrophoresis, laboratory techniques; The Coomassie brilliant blue R250 staining and destaining procedure given in the booklet was followed. The pigments, partially purified by gel filtration, were studied as follows: 1. Pigments were examined in PAGE both in gradient gel slabs run for 2000 vhr and in rods (T = 8.4, C = 2.9). Pigmented bands of interest (including ~-crustacyanin) were excised, sliced longitudinally and incubated (a) for 1.5 hr at 37°C in 27~, SDS-0.125 M tris HCI, pH 6.8, - 5 % sucrosebromophenol blue (BPB)(10/~g/ml), with and without 5°~ mercaptoethanol (ME), (b) for 1.0hr at 37'C in 6 M urea 0.125 M tris-HCl, pH 6.8, - B F B (10/lg/ml). The slices were then placed both longitudinally and vertically on the spacer gel of the appropriate SDS-acrylamide gradient or 6 M urea slab gel (T = 8.4, C = 2.9), covered with the incubation solution and electrophoresis performed at 100V. A molecular weight calibration kit (M.W. 14.4 x 103-94 × 103; Pharmacia) was treated as in (a), above, and run on a separate gel slab. Gels made with and without SDS were stained as in Bordier & Crettol-Jarvinen (1979), and those containing urea by the method of Cleveland et al. (1977). 2. Yellow protein ()'max 409 nm) was focused as a fairly broad band near the bottom (surface pH 4.6-4.8) of IEF rods. The bands were excised, incubated with 0.068 M tris HC1, pH 6.8-5~o sucrose-BFB (10 tlg/ml) for 45 min at 20°C. The pieces were then placed onto the spacer gel ol rods (T = 8.4, C = 2.9), covered with incubation solution and PAGE performed with the discontinuous buffer system at 30 V overnight. The yellow band was excised, sliced longitudinally and treated as for slices in (1), above. 3. Chromatofocusing of yellow protein (2,,ax 409 rim), partly purified by Sephadex G200, on polybuffer exchanger PBE T M 94 was carried out as described in the Pharmacia chromatofocusing kit booklet, with polybuffer 74 as eluant.
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Fig. l. Gel filtration elution profile of lobster pigments on Sephadex G200. The sample was the pigment eluting in DEAE cellulose chromatography with 1 M KCI 0.05 M phosphate buffer, pH 6.9 (see Methods section), following passage of 0.25 M phosphate buffer, pH 6.9. The column (75 x 3.2cm) was equilibrated with the eluant, l M KCI 0.05 M phosphate buffer, pH 6.9, in the dark at 3C. Sample volume: 3 ml. Fraction volume: 3 ml. Flow rate: 10 ml/hr. Curves represent the extinctions of fractions at 400 nm, , and at 630 nm,- . . . . . ; the values of the ratio of extinctions at 400 and 280nm (E40o/280) are given, . Inset: rechromatography of pooled fractions B, yellow pigment (2 ...... 409 nm).
Following passage of the polybuffer the strongly adsorbed pigment was partly eluted with 0.5 M KCI. The pigment was concentrated by ultrafiltration, dialysed against 0.068 M tris HC1, pH 6.8, and subjected to discontinuous PAGE in rods, or alternatively, gradient gel slabs for 2000vhr. Pigment bands of interest were excised and treated as for slices described in (1), above.
0.8 60
It is clear from the gel filtration studies (Figs. 1 3) that, in addition to yellow protein (2 ...... 409nm), extracts of lobster carapace contain other pigments of larger molecular size absorbing in the 400 nm region. Pigments eluting prior to the yellow protein on columns of Sephadex G200 (Fig. 1) were partially resolved on columns of Agarose 4B (Fig. 2) and subsequent r e c h r o m a t o g r a p h y on columns of Sephadex G200 (Fig. 3) into the following fractions: a minor red pigment, excluded from Agarose 4B, with 2 ...... at 398 4- 1 nm, 470 _+ 2 nm and 620 4- 5 nm of relative extinction 1.0/0.8/0.35; a fraction (Pigment Yl) larger in size than ;¢-crustacyanin, of Stokes radius 130 A, with ,:,...... at 398 4- 1 n m and 470 4- nm of relative extinction 1.0/0.7; a fraction (Pigment Y j smaller in size than =-crustacyanin, of Stokes radius 60/L with )....... at 401 _+ 1 nm and an inflexion at 470 nm. Pigments Y1 and Y2 were incompletely resolved from residual ~-crustacyanin and from each other. In gradient gel electrophoresis Pigment Yt moved as a red yellow band and Pigment Y2 as a yellow zone of lower and greater mobility, respectively, than ~-crustacyanin (Fig. 4). Both pigments gave predominantly two subunits in S D S - P A G E (Fig. 5), identical in size to those of =-crustacyanin; the proteins did not dissociate in 6 M urea.
120
180 240 Effluent volume, ml
300
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Fig. 2. Gel filtration elution profile of pigment fraction A (see Fig. 1) on Agarose 4B. The column (75 x 3.2 cm) was equilibrated and run as stated in Fig. 1. Curves represent the extinction of fractions at 400 nm, , and at 630 nm, . . . . . . . Values of E40o,,280 are given,
The best preparations of yellow protein (2 ...... 409 nm) obtained in gel filtration had a ratio of absorptions at 409 and 280 n m (E409/280) of 3.4. The partly purified protein eluted in a symmetrical but b r o a d band (Fig. 1 inset) with a Stokes of ca. 37,&. The behaviour of the protein in gel electrophoresis is unusual. In PAGE, both with continuous (tris borate-EDTA)* and discontinuous buffer systems, the pigment showed considerable spread and uneven protein staining within the pigment region (Fig. 4). This
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Fig. 3. Gel filtration elution profiles of pigment fractions C and D (see Fig. 2) on Sephadex G200. (A) Pigment fraction D. (B) Pigment fraction C. The column (75 x 3.2 cm) was equilibrated and run as stated in Fig. I. Curves represent the extinction of fraction at 400 nm, - , and at 630 nm, . . . . . . . Values of 54-oo128o are given, . Pigment fractions Y~ and Y2 are referred to in the text.
A study of the yellow astaxanthin-proteins of lobster carapace
245
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Fig. 4. Electrophoresis of yellow protein (2,,ax 409 nm) following gel filtration on Sephadex G200 and of pigment fraction A (Fig. 1). Yellow protein (2max 409 nm): (a) PAGE (T = 8.4, C = 2.9). (b) PAGE (T = 10.9, C = 2.9). (c) IEF, pH 3-10. (d) PAGE (T = 8.4, C = 2.9) of excised pigment zone following IEF. (e) PAGE in gradient gel for 2000 vhr. The zone of yellow protein is indicated between the arrows. Pigment fraction A (Fig. 1): (f) PAGE in gradient gel for 2000vhr. YI, pigment Y1; Y2, pigment Y2; ct,~-crustacyanin.
occurred also in IEF at pH 3-10, in PAGE following IEF, and in gradient gel electrophoresis run for 2000vhr to concentrate the protein at its exclusion point. Two groups subunits were evident when the gel slice of yellow protein obtained in PAGE or IEF or gradient PAGE was subjected to S D ~ P A G E in an orientation perpendicular to the direction of mobility in the former gels (slice horiz. Fig. 5). Subunits (Type 1) of an identical size to those of ct-crustacyanin (1.9 x 104 and 2.2 x 104) were present fairly evenly across the slice region and, in some gels, a component of greater mobility, of apparent molecular weight 1.3 x 104. In addition, slanting protein bands (usually two), of highest molecular size for the yellow pigment of lowest mobility in the first gel, covered the pigmented zone of the slice. These bands were resolved into 4 components (Type II subunits) of apparent molecular size 5 x 104-8 x 104, not always present in the same relative proportions, when the electrophoresis was performed with the slice in an orientation parallel to that of the first run (Slice vert. Fig. 51. The electrophoresis pattern was unaltered when slices were treated with mercaptoethanol. In 6 M urea-PAGE, slices run in a perpendicular orientation to that in the first direction (PAGE, IEF or gradient PAGE) similarly showed slanting protein bands, together with the subunits common to ct-crustacyanin. The latter on occasions appeared only as minor components (Fig. 5). The protein was eluted from polybuffer PBE TM 94 with an altered absorption spectrum. The fraction, with 2 .... at 411 nm and a minor inflexion at 470 nm, showed a stronger absorption at 280 nm (with a value
of E411/280 of 1.5) than the starting sample. It gave both Type I and Type II subunits in SDS-PAGE (Fig. 5). DISCUSSION
Recent interest in the distribution of astaxanthinproteins within lobster carapace has focused attention to their possible photobiological function (Salares et al., 1977; Mackenthun et al., 1979). It has been proposed that the yellow protein at the outermost layer of the endocuticle acts as a light receptor passing energy inwards to pigments of bathochromically shifted spectra (Salares et al., 1977; Mackenthun et al., 1979). Photoacoustic spectroscopy (PAS) of carapace reveals an anisotropic distribution of pigments with a steady shift in absorption maximum from 450 to 625 nm, from outer to inner parts of the endocuticle, together with pigments hypsochromically shifted in spectra at the outer (~'max 425 nm) and inner surface (2ma x 390nm). Infrared resonance Raman spectroscopy has provided evidence that pigments with absorption maxima at even longer wavelength than ct-crustacyanin may be present in the carapace (Nelson & Carey, 1981). It is uncertain whether additional pigments to those already identified (ct- fl- and ),-crustacyanins and yellow protein (~'max 409 nm)) are yet to be isolated or whether interactions of the latter proteins within the carapace can account for the above observations. The absorption spectra of pigment fractions Yz and Yz isolated in the present study cover the lower wavelength absorption within the endocuticle apparent in PAS. It would be facile to conclude that pigment Y1
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Fig. 5. Electrophoresis of yellow protein (2 .... 409 nm) in gels containing SDS or 6 M urea. (aF(n) SDS- PAGE in gradient gels. (aF(c) excised pigment zone following PAGE ( T = 8.4, C = 2.9) (Fig. 4a): (a) slice horiz., (b) slice horiz., treated ME, (c) slice vert. Apparent molecular sizes of Group II subunits (see text) derived from a plot of RBVB VS log molecular weight: (1) 7.9 x 10 4 (2) 6.6 x 104 (3) 5.5 x 104 (4) 4.9 x 104. (d) Yellow protein purified by chromatofocusing and PAGE in gradient gel for 2000 vhr: excised pigment zone, slice vert. (eF(fl pigment Ya (e) and YI (f) excised from gel, Fig. 4(f). (g) e-crustacyanin excised from PAGE gel; subunit nomenclature (C2, C~, A,, As and A3) as in Quarmby et al. (1977). (h) Following IEF, pigment zone excised (Fig. 4c) and subjected to PAGE [T = 8.4, C = 2.9). Pigment zone excised from PAGE gel (Fig. 4d), slice vert. (i) As in (e). (j) As in (h), slice horiz. (k) Yellow protein purified by chromatofocusing and PAGE (T = 8.4, C = 2.9). Pigment zone excised from PAGE gel, slice vert. (1) As in (h), slice horiz., treated ME. (m) Pigment zone following PAGE in gradient gel for 2000 vhr {Fig. 4el, slice horiz. (n) As in (e). (oh(t) 6 M urea PAGE (T = 8.4, C = 2.9). (o) As in (m). (p) As in (e). (q) As (m), slice vert. (r) As in (j). (s) As in (h). (t) As in (e).
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A study of the yellow astaxanthin-proteins of lobster carapace
247
A heterogeneous mixture of yellow proteins, is located at the outer part of the endocuticle and 7 × 104-10 × l04 in molecular size, is consistent with pigment Y2 at the inside. It is not possible to decide, the molecular weight studies (Buchwald & Jencks, however, whether either pigment is native to the cara1968) of this fraction. pace or is formed during the extraction procedure, since crustacyanin denatures to pigments of similar size and spectrum at high pH (Jencks & Buten, 1964; Buchwald & Jencks, 1968), a condition which may REFERENCES arise during extraction. The unstable yellow protein (2 .... 409 nm) has a BORDIER C. & CRETTOL-JARVINENA. (1979) Peptide mapping of heterogeneous protein samples, d. biol. Chem. molecular weight of ca. 90,000 and a minimum mol25,1, 2565-2567. ecular weight, based on the astaxanthin content, of BUCHWALD M. & JENCKS W. P. (1968) Properties of the 4400 (Buchwald & Jencks, 1968). The protein comcrustacyanins and the yellow lobster shell pigment. Bioponent of the pigment thus represents a weight of chemistry 7, 844-859. about 77,000, assuming 20 astaxanthin prosthetic CHEESMAND. F., ZAGALSKVP. F. & CECCALDIH. J. (1966) groups. The amino acid composition is distinct from Purification and properties of crustacyanin. Proc. R. Soc. :t-crustacyanin (Buchwald & Jencks, 1968) and from BI64, 130-151. the individual subunits of this protein (Quarmby et CLEVELAND D. W., FISHER S. G., KIRSCHNER M. W. & al., 1977). The heterogeneous nature of this yellow LAEMMLI U.K. (1977) Peptide mapping by limited proprotein component, regardless of the method used to teolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. biol. Chem. 252, 1102 1106. separate the pigment, is evident in the present study. HJERTEN S. (1962) Molecular sieve chromatography on Two types of subunit, based on molecular size, may polyacrylamide gels, prepared according to a simplified be distinguished. The subunits of Type II, of apparent procedure. Archs. Biochem. Biophys. Suppl. I, 147-t51. size 50 × 103 80 × 103, must be responsible for the JENCI,:SW. P. & BUTENB. 0964) The denaturation of crushigh glutamate content (Buchwald & Jencks, 1968) of tacyanin. Archs. Biochem. Biophys. 107, 511 520. the pigment. The proteins of Type I, common to KUHN R. & KOHN H. (1967) Quat~irstruktur und farbe yon ~-crustacyanin, account for the immunological crosscrustacyanin. Eur. J. Biochem. 2, 349-360. reactions between the yellow protein and the crustaLAEMMLIU. K. & FAVREM. (1973) Maturation of the head of the bacteriophage T,,. I. DNA packaging events. J. cyanins (Buchwald & Jencks, 1968). molec. Biol. 80, 575-599. The polydisperse behaviour of the pigment in the MACKENTHUN M. t., TOM R. D. & MOORE T. A. (1979) electrophoretic investigations may be explained if the Lobster shell carotenoprotein organisation in situ quaternary structure is variable and consists of pairs explored by photoacoustic spectroscopy. Nature, Lond. of subunits, each pair containing a peptide from each 278, 861-862. of the two types of subunit. The relative proportions NELSONW. H. & CAREVP. R. (1971) Infrared excited resoof subunits within each class in any given preparation nance Raman Spectra of lobster shell pigments in situ. d. would then depend on the particular mixture of pigRaman Spectrosc. In press. ment molecules present. It would seem reasonable to QUARMBYR., NORDEND. A., ZAGALSKYP. F., CECCALDIH. suppose that the number of astaxanthin in carotenoid J. & DAUMASR. (1977) Studies on the quaternary structure of the lobster exoskeleton carotenoprotein, crustamicelles of pigment molecules of different subunit cyanin. Comp. Biochem. Physiol. 56B, 55-61. composition may also be variable (about the mean of 20), unless the smaller group II subunit (5 × 10 4) is SALARESV. R., YOUNGN. M., BERNSTEINH. J. & CAREYP. R. (1977) Resonance Raman spectra of lobster shell caroable to associate with the same size of carotenoid tenoproteins and a model astaxanthin aggregate. A possmicelle as the larger (8 × 104 ) subunit; it would ible photobiological function for the yellow protein. Bioappear from the electrophoretic results that the subchemistry 16, 4751-4756. units of lower molecular size (Type I) are more readily ZAGALSKVP. F. & HERRINGP. J. (1977) Studies of the blue lost from the association during purification proastaxanthin-proteins of Velella velella (Coelenterata: cedures and may therefore not play so important a Chondrophora). Philos. Trans. R. Soc. London. Ser. B. 279, 289-326. role in stabilisation of the carotenoid micelle.