From the Dipartimento di Biologia and Centro di
Ricera Interdipartimentale per le Biotechnologie Innovative
Biotechnology Center, Università di Padova, Via Ugo Bassi 58/B,
35131 Padova, Italy, the ¶ Dipartimento di Scienze e Tecnologie
Avanzate, Università del Piemonte Orientale Amedeo Avogadro,
Corso Borsalino 54 I-15100 Alessandria, Italy, the
Dipartimento di Biologia and Consiglio
Nazionale delle Ricerche Center for the Study of Metalloproteins,
Università di Padova, via Ugo Bassi 58/B, 35131 Padova, Italy,
and the
Department of Biochemistry, University of Antwerp UIA,
Antwerp, Belgium
Received for publication, August 2, 2000, and in revised form, April 2, 2001
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ABSTRACT |
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Annelid hemoglobins are organized in a
very complex supramolecular network of interacting polypeptides, the
structure of which is still not wholly resolved. We have separated by
two-dimensional electrophoresis the 4-MDa chlorocruorin of
Sabella spallanzanii and identified its components by
amino-terminal sequencing. This work reveals a high rate of
heterogeneity of constituent chains in a single animal as well as in
the Sabella population. Using a cDNA library prepared
from the hematopoietic tissue of this worm, we have isolated and fully
sequenced most globin and linker cDNAs. The primary structure
features of these polypeptides have been characterized by comparison
with model globin and linker sequences.
Chlorocruorins (Chls)1
are giant extracellular oxygen-binding heme proteins found in four
marine polychaete families. They contain an altered heme with a formyl
group substituting the canonical three-vinyl group and consequently
appear as greenish red (1). The Chls have in electron micrographs the
hexagonal bilayer (HBL) appearance and size characteristic of annelid
and vestimentiferan extracellular Hbs, a similar sedimentation
coefficient of ~60 S (molecular mass ~3, 600 kDa), and an
abnormally low iron content of 0.23 weight % (2-6). The HBL Hbs,
including the Chls, have almost identical three-dimensional structures
as determined by cryoelectron microscopy at about 3-nm resolution
(7-11).
All HBL Hbs and Chls are composed of two type of chains,
heme-containing 16-17-kDa globin chains and nonglobin linker chains of
25-32 kDa in an approximate 2:1 molar ratio (1). The structural hierarchy within the HBL Hb of Lumbricus terrestris was
determined recently by x-ray diffraction analysis at 5.5 Å resolution
(12). This Hb, composed of 144 globin and 36 linker chains, is arranged in dodecameric substructures. Twelve trimeric linker complexes project
triple-stranded helical coiled-coil "spokes" toward the center of
the complex; interdigitation of these spokes seems crucial for
stabilization. The resulting complex of linker chains forms a scaffold
on which 12 Hb dodecamers assemble.
The nature of the disulfide-bonded globin subunits composing the
dodecameric structures is different among the different classes. Monomers and disulfide-bonded trimers or monomers and disulfide-bonded dimers are found in oligochaete/polychaete and leeches/vestimentiferans Hbs, respectively (5, 13, 14). In contrast the Eudistylia vancouverii Chl (15, 16) consists of two types of globin
subassemblies, a dodecamer formed by the noncovalent association of
three disulfide-bonded trimers and a tetramer formed by the noncovalent
association of disulfide-bonded dimers.
In contrast to annelid Hbs, in which a wealth of sequence information
of globin chains is available, only a single primary structure of a Chl
globin chain has been published (17). Globin chain E of
Sabellastarte indica shows 27-49% sequence identity with the annelid globin chains. Five cysteines, crucial for the subunit
formation, are present. Two adjacent cysteines just preceding A1 and
one at position H11 are conserved as in all annelid globin chains of
type I (18). Two other cysteines occur at position E8 and within
the corner between the G and H helices, as frequently seen in
other annelid Hbs as well (19).
The Chl of Sabella spallanzanii, a marine fan worm
polychaete formerly known as Spirographis spallanzanii, was
chosen as a model molecule to identify the set of polypeptides that
form the complete protein. The globin and linker polypeptides that
compose its supramolecular structure have been resolved by
two-dimensional gel electrophoresis. Three globin and three linker
mRNAs and some variants have been isolated from S. spallanzanii cDNA libraries by screening with specific
antibodies and have been completely sequenced and characterized.
Purification and Gel Separation of S. spallanzanii Globin and
Linker Chains--
Live specimens of S. spallanzanii were
collected in the Bay of Napoli. When Chl was prepared from pools of
animals, the collected hemolymph was first centrifuged (10 min at
1,000 × g) and then frozen at
One- and two-dimensional electrophoresis (2-DE) were performed
according to Fling and Gregerson (20) and Görg et al.
(21). The first dimension was isoelectric focussing on a 4.0-7.0
Immobiline pH gradient. A 15% polyacrylamide/SDS slab gel
electrophoresis was used as second dimension.
Protein Sequencing--
Globin and linker chains were separated
by 2-DE, and the band pattern was transferred to a polyvinylidene
fluoride membrane (Millipore). Selected spots were sequenced in an
Applied Biosystems ABI 471-B sequencer operating as recommended by the manufacturer.
cDNA Library Construction--
The monolayer hematopoietic
tissue of S. spallanzanii (22) was prepared from living
animals, immediately frozen in liquid nitrogen, and kept at Production of Anti-Chl Antibodies--
Purified Chl was
separated into its components by SDS-polyacrylamide gel electrophoresis
(20), and the gel was stained with acid-free Coomassie. Three main
bands were cut out from the gel and electro-eluted as described
previously (25). After checking the purity by a second
SDS-polyacrylamide gel electrophoresis run, the purified proteins were
emulsified with complete Freund's adjuvant and injected into rabbits.
Animals were booster-injected every second week over a period of 2 months. Blood was collected, and the serum was separated by
centrifugation and stored at Screening and Sequencing of Sabella Globin and Linker cDNA
Clones--
In a first round of screening, 100,000 bacteria colonies
were grown on 10 nylon filters laid on square Luria Bertani broth plates (27). The colonies were then replicated twice on nitrocellulose filters and re-grown on Luria Bertani broth plates containing isopropyl
Sequence Analysis--
Several programs, available at the ExPASy
molecular biology server, were used to analyze the new
sequences. The ORFs codified by the cDNAs were obtained using the
Translate program. Then the ORFs were manually aligned to the
polypeptide amino termini sequenced during this work or with globins
and linkers available from the literature and data bases to identify
the export signal peptides. Successively, the pI and molecular mass
were computed from the cDNA sequences with the program Compute
pI/Mw and compared with the experimental data obtained from the 2-DE
gel analysis. The pI and Mw measured with the two protocols are
perfectly comparable. Amino acid compositions of the mature
polypeptides were determined with the program ProtParam. Finally, the
linker chains were aligned using the ClustalW program (28), and the
globins were manually aligned according to the tertiary structure
template of invertebrate globins (19).
Determination of the Chl Polypeptide Components--
A
representative 2-DE separation of the Chl extracted from a single
specimen is shown in Fig. 1A.
A complex pattern of multiple spots is clearly detectable. They can be
divided into three groups: heavy linkers (molecular mass ~35 kDa),
light linkers (molecular mass ~31 kDa), and globin chains (Glb)
(molecular mass ~14.4 kDa). This pattern is comparable with that
obtained for the Chl of E. vancouverii, in which two groups
of linkers (L1a-f and L2a-d, 10 chains) and six
globin chains where detected by electron spray ionization mass
spectrometry (16). Although the solution of the
three-dimensional structure of the HBL Hb of L. terrestris offers a splendid explanation for the structural
hierarchy within the molecule, no rationale is presented for the globin
and linker chain multiplicity (12). The observed high cooperativity of
the HBL Hbs (Hill coefficient n50 > 3) (29-32) as
well as the aggregation into trimers, tetramers, and dodecamers definitively need different globin types (1) (a,
b, c, and d in L. terrestris). The formation of the coiled coils in the linker
scaffold complex also probably needs structurally different linker
chain types (1) (L1-4 in L. terrestris).
A comparison of the globin and linker chains of a single animal (Fig.
1A) with that of a pool of 500 S. spallanzanii
specimens (Fig. 1B) reveals a high degree of heterogeneity.
Sixteen spots in the range of ~31 kDa and thirteen in the range of
14.4 kDa are clearly distinguishable. Amino-terminal sequencing of the major spots and alignment with published sequences confirm their identification as linkers and globin chains (Table
I). There are three possible explanations
for this heterogeneity: (i) the described variations (see below)
suggest the presence of multiple copies of the same gene such that
allelic as well as nonallelic variations can occur, (ii)
post-translational modifications, or (iii) artificial modifications
might be induced by the extraction and separation procedures used (21).
It should be considered however that in invertebrates as well as in
vertebrates, multiple copies of globin genes are a rule rather than an
exception (33-35). Therefore this multiplicity could most likely be
explained by the necessity to synthesize huge amounts of the oxygen
carrier (35). A final conclusion on the exact number of globin and
linker chains in S. spallanzanii must wait for a careful
analysis of the Chl of single animal by electron spray ionization mass
spectrometry.
Characterization of Specific Antibodies against Chl
Components--
For the molecular cloning of globin and linker
cDNAs, we produced specific antibodies as described under
"Materials and Methods." Immunological tests on Chl Western blots
reveal that the produced antibodies are efficient and specific for the
detection of the polypeptides for which they were developed (Fig.
2). No cross-reaction was found between
antibodies for globins and linkers. Some cross-reaction, however, was
observed for the two classes of linkers (heavy and light) with the
corresponding antisera, probably because of the high level of
similarity among the members of the two classes.
Cloning and Sequencing of Sabella Globin and Linker
cDNAs--
A cDNA library was constructed using mRNA
extracted from the hematopoietic tissue of S. spallanzanii.
Immunological screening detected many recombinant clones that were
sequenced. Six complete cDNAs, coding for three globin and three
linker chains, have been identified and are presented in Fig.
3, A and B,
respectively. Each cDNA represents the consensus sequence of at
least three independent clones except for Glb3, for which only two
positive clones were obtained (see "Materials and Methods"). The
main features of the cDNA sequences are summarized in Table
II.
The ORF codified by the Glb1 cDNA can be identified as the
polypeptide J in 2-DE (Fig. 1B) because the
corresponding amino-terminal sequences are identical. Two variants for
Glb2 cDNA were obtained that differ for two transversions in the
coding region. The first (G versus T) is placed in the third
base of the codon 66 and does not change the coded amino acid (Leu).
The second (A versus C), however, produces a variation of
the ORF (codon 92) with the presence of Ala instead of Asp in six of
seven independent Glb2 cDNA clones. The signal peptide of Glb2 was
identified by aligning the amino-terminal sequences obtained from the
Glb B and C 2-DE spots (Fig. 1B; Table I). The cDNA for Glb3 is about 200 bases shorter than the cDNAs for Glb1 and Glb2. The signal peptide was identified aligning the ORF
with the amino-terminal of the Glb D polypeptide. For this
cDNA we found also some variants that differ both in the coding and
in the 3'-noncoding regions. The C
The cDNAs of L1 and L2 share 99% identity and code for the
same ORF (Fig. 3B). They are identical in the 5'-noncoding
regions, whereas some transitions and transversions are accumulated
in the coding and in the 3'-noncoding regions. The most important differences are a six-base deletion (5'
The amino acid compositions of the globin and linker chains deduced
from the cDNA sequences were computed using the program ProtParam.
The data are summarized in Fig. 4. Glb1
and Glb2 have a rather similar composition even though they show some
peculiar differences (e.g. Glu is more abundant in Glb2).
The amino acid composition of Glb3, however, differs more strikingly
from the other two globins. This is particularly evident for Ser
residues. The percentage of Ser in Glb3 is twice that in Glb1 and Glb2. As mentioned above, L1 and L2 cDNAs code for an identical
polypeptide that differs in composition from L3 (e.g. the
percentage of Ser in L1 and L2 is twice that in L3, whereas Lys instead
is two times more abundant in L3). Both globin and linker chains show a
high percentage of negatively charged residues that explains the low pI
calculated for all the chains (Table II).
Sequence Alignment and Characterization--
To characterize the
primary structure of the Sabella globin chains, we have
aligned them with the annelid, pogonophoran, and vestimentiferan globin
sequences available in our data base including a globin chain from the
Chl of S. indica and globin references (17-19). A
representative selection of this alignment is presented in Fig.
5. The alignment is unambiguous because
of the presence of the globin landmarks A12-Trp, C2-Pro, CD1-Phe,
E7-His, F8-His and H8-Trp. All three novel globin chains fit the
nonvertebrate globin template quite well, resulting in low penalty
scores (19). Glb3 has a hydrophobic residue at the surface positions A6
and CD2. Because similar hydrophobic substitutions occur in other annelid globin sequences, it might be that they represent specific adaptations for the aggregation into high molecular mass complexes. No
specific adaptations can be localized to harbor the formyl group on the
heme ring. The positions of cysteine residues in annelid globin chains
are strictly conserved because of their role in the formation of
disulfide-bonded subunits (18). On the basis of the pattern of these
residues, two types of chains can be distinguished. Type I has
absolutely conserved cysteines at positions NA2 and H11 and a less
conserved one at position GH4. Type II displays the same pattern
with an additional cysteine at position NA1. As such, Glb3 can be
classified as type I and Glb1 and Glb2 can be classified as type II. In
all annelid-like globin chains studied thus far, the cysteines at
positions NA2 and H11 are involved in an intrachain disulfide bridge
linking the NA terminus to the H-helix and leaving the two other
cysteine residues free for the formation of inter-chain bridges (12, 16). As such, based on the alignment it can be concluded that the
globin chains of S. spallanzanii are similar to the other annelid, pogonophoran, and vestimentiferan globin sequences. Therefore, the Sabella globin primary structure is not sufficiently
informative to explain how these Hbs have acquired the possibility of
harboring the modified heme group.
A similar alignment was carried out for the linker sequences translated
from the cloned cDNAs with two other linkers of
Lumbricus and Lamellibrachia (Fig.
6). The most remarkable feature of the linker chains, including these of S. spallanzanii, is a
conserved 38-39-residue segment containing a repeating pattern of
cysteinyl residues
((Cys-X5-6)3-Cys-X5-Cys-X10-Cys)
(36-38). This pattern is identical with the cysteine-rich repeats of
the ligand-binding domain of the low density lipoprotein receptors of
man and Xenopus laevis. The sequence Asp-Gly-Ser-Asp-Glu,
characteristic of the low density lipoprotein receptor repeats, is less
conserved in S. spallanzanii L1, L3, and
Lamellibrachia LAV1 than in L. terrestris L1.
Nevertheless, the pattern Asn-Gly-X-Asp-Glu is easily
recognizable in the S. spallanzanii linker sequences (38).
Two other cysteines at positions 129 and 228 are also conserved
together with a Leu, a Gly, and a Tyr residue at positions 31, 122, and
182, respectively. The suggestion that linker chains resulted from gene
duplication of a heme-containing chain with a three exon/two intron
structure and that the first exon of domain 1 and the last exon of
domain 2 have been lost during evolution can not be confirmed (37). It
is more likely that the cysteine-rich motif of the low density lipoprotein receptor and linker chains represents a multipurpose protein-binding unit of ancient origin that has been incorporated into
diverse unrelated proteins by the process of exon shuffling (38).
In this paper we have presented the primary structure of three globin
and three linker chains composing the Chl of S. spallanzanii and thereby significantly increase the number of annelid, pogonophoran, and vestimentiferan sequences available today. A detailed evolutionary analysis of our data in combination with that available from the literature is presented in an accompanying paper (39).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
20 °C in 0.1 M Tris/HCl, pH 7.0, 35 mM CaCl2,
and 20% sucrose. Tablets of the Complete protease inhibitor kit
(Roche) were added to prevent proteolytic activity. The Chl was
sedimented by ultracentrifugation (3.5 h at 300,000 × g) at 4 °C, and the obtained pellet was resuspended in
the same buffer and stored at
20 °C. The Chl concentration was
determined using E
80 °C.
The extraction of total RNA was performed according to Chomczynsky and
Sacchi (23) using the tissues of a total of seven specimens.
Poly(A)+ mRNA was purified by oligo(dT) affinity
chromatography (24). The first strand cDNA was synthesized with an
oligo(dT)-NotI primer using the Copy kit (Invitrogen). The
double-stranded blunt-ended cDNA was ligated to BstXI
nonpalindromic adaptors, NotI-digested, and finally
directionally cloned into a BstXI-NotI-cut
pcDNAII plasmid vector (Invitrogen). The recombinant vectors were
electroporated into Escherichia coli strains Inv
F' and
Top10F' (Invitrogen).
80 °C. As controls, sera were
collected from the same rabbits before the immunization protocol. The
specificity of the different antisera was checked by immunoblotting
according to standard protocols (26).
-D-thiogalactopyranoside as an inducer for the
expression of the recombinant proteins. The clones containing the
globin and linker cDNAs were detected by immunological screening
using the specific antibodies at a dilution of 1:1,000. Two rounds of subscreening were performed to select single positive clones. Both
strands of the cDNA inserts were sequenced by a primer-walking strategy using the fluorescent BigDyeTM terminators chemistry (PE Biosystems), and the sequencing reactions were analyzed on an ABI-377
automated DNA sequencer (PE Biosystems). The sequences were assembled
using the SeqMan II program from the Lasergene software package
(DNAStar, Madison, WI). Full-length sequences of linker 3 and globin 1 were completed by 5' and 3' rapid amplification of cDNA ends on the
S. spallanzanii cDNA. A primer designed on BstXI adaptor was used for the 5' rapid amplification of
cDNA ends, whereas the oligo(dT)-NotI was used as primer
for the 3' rapid amplification of cDNA ends reaction. One µl of
adaptor-ligated cDNA was mixed with 200 µM dNTPs, 200 nM primers, 2.5 units of AmpliTaq GOLD (PerkinElmer Life
Sciences), and 1.75 mM MgCl2. After the
activation of AmpliTaq GOLD at 94 °C for 15 min, 35 cycles of
amplification were performed using the following steps: denaturation at
94 °C for 30 s, annealing at 50 °C for 1 min, and extension
at 72 °C for 1 min. The same protocol was adopted for the isolation
of globin 3 cDNA, because immunoscreening identified no positive
clones. In this case, a degenerated reverse primer coupled to the
BstXI adaptor primer was designed on the amino terminus of globin D. A 180-base pair fragment was obtained and sequenced. On this sequence two nested forward primers were designed for reaching the 3' end of mRNA using the oligo(dT)-NotI
as reverse primer.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
2-DE patterns of S. spallanzanii
chlorocruorin. A, representative 2-DE pattern of
Chl from a single S. spallanzanii specimen. Two groups of
components could be identified. The low molecular mass group consists
of at least 10 different globin polypeptides. In the higher molecular
mass linker group a total of eight spots could be detected.
B, 2-DE pattern of Chl pooled from 500 S. spallanzanii specimens. A higher number of spots are
detectable. A-M, globin spots; a-p, linker
spots; Mw, molecular mass of marker proteins.
Primary structure of Sabella globins and linkers
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Fig. 2.
Characterization of antibodies against
Sabella Chl components. Chl chains were separated
by SDS-polyacrylamide gel electrophoresis, blotted, and revealed by
Ponceau red (lane A), pre-immune sera (lanes B,
D, and F), and immunostaining with antibodies
raised against heavy linkers (LH), light linkers (LL),
and globins (lanes C, E, and G,
respectively). Mw, molecular mass of protein
standards.
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Fig. 3.
cDNA sequences and deduced ORFs of the
S. spallanzanii Chl components. A,
globin chains; B, linker chains. Signal peptides and
polyadenylation sites are underlined. ORF amino acids and
variant nucleotides are in bold.
Main features of linker and globin cDNAs of S. spallanzani
chlorocruorin
T transition in the coding region
(codon 42) produces Pro-Leu variants that have the same pI but vary
slightly in molecular mass. One transversion and two transitions are
also present in the 3'-noncoding regions of these two variants.
GAATA
3') and the
insertion of a single T in L2. These two genes could have originated in a very recent gene duplication, because the observed differences are
regularly present in all the independent clones. The amino-terminal amino acid sequences deduced from L1 and L2 cDNAs match with the amino-terminal sequences obtained, at the protein level, from the spots
f and g in the 2-DE of Chl components (Fig.
1B; Table I). The cDNA for L3 is markedly shorter than
those for L1 and L2; nevertheless it codes for an ORF that is two amino
acids longer. No amino terminus was available to determine the signal
peptide of L3. However, a putative signal can be identified by aligning the ORF with the amino terminus of Linker LAV1 of
Lamellibrachia sp. (36).
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Fig. 4.
Amino acid composition of S. spallanzanii chlorocruorin globin and linker chains.
Data are expressed as percentages. For Glb2 and Glb3, the data refer to
the Ala and Leu variants, respectively.
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Fig. 5.
Alignment of globin chains. The
sequences have been aligned in the coding portions using as a reference
(19) the tertiary structure template of invertebrate globins (Globin
fold). Physeter Mb (P02185), Physeter catodon
myoglobin; Homo Hb (P01922), Homo sapiens
-chain; Homo
Hb (P02023), Homo sapiens
-chain; Lumbricus d1 (U55073), L. terrestris
Hb d1; Sabella Glb3 (AJ131285), S. spallanzanii
globin 3; Lumbricus IV (P13579), L. terrestris Hb
IV; Sabellastarte E (D58418), S. indica globin;
Sabella Glb1 (AJ131283), S. spallanzanii globin
1; Sabella Glb2 (AJ131284), S. spallanzanii
globin 2 are shown (the accession numbers from the Swiss-Prot, NCBI,
and EMBL data banks are in parenthesis). Black bars
indicate the position of amino acids that are important for the globin
fold, and the gray bars indicate the cysteine residues that
characterize the two groups of annelid globins (see text for
details).
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Fig. 6.
Alignment of linker chains. Sabella L1
(AJ131900), S. spallanzanii linker 1;
Lumbricus L1 (A46587), L. terrestris linker 1;
Lamellibrachia LAV1 (P16222), Lamellibrachia sp.
linker LAV1; Sabella L3 (AJ131286), S. spallanzanii linker 3 are shown (the accession numbers from the
Swiss-Prot, NCBI, and EMBL data banks are in parentheses).
Black bars indicate the position of invariant amino acids,
and the asterisks underline the conserved cysteine-rich
segment (see text for details).
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ACKNOWLEDGEMENTS |
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We thank Dr. Lucio Cariello Director of the Stazione Zoologica "A. Dhorn" (Napoli, Italy) for providing the specimens of Sabella spallanzanii and Drs. Silvia Trevisan, Beniamina Pacchioni, and Rosanna Zimbello for help in sequencing. Marie-Louise Van Hauwaert is acknowledged for skillful technical assistance.
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FOOTNOTES |
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* This work was supported in part by the Italian Ministry of University and of Scientific and Technological Research MURST Grant-Cofinanziamento Protocol 9805192993-002 (to A. G.-M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AJ131283 (globin 1), AJ131284 (globin 2), AJ131285 (globin 3), AJ131900 (linker 1), AJ131901 (linker 2) and AJ131286 (linker 3).
§ Both authors contributed equally to this work.
** Post-doctoral fellow of the Fund for Scientific Research-Flanders (FWO).
§§ To whom correspondence should be addressed. Tel.: 390-4982-76221; Fax: 390-4982-76280; E-mail: lanfra@cribi.unipd.it.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M006939200
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ABBREVIATIONS |
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The abbreviations used are: Chl, chlorocruorin; HBL, hexagonal bilayer; 2-DE, two-dimensional gel electrophoresis; ORF, open reading frame; Glb, globin; L, linker; pI, isoelectric point.
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