of chemistry vol. no issue may 6502-6517,1988 1988 the u ... · the journal 0 1988 by the american...

THE JOURNAL 0 1988 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 263, No . 14, Issue of May pp. 6502-6517,1988 Printed in U. S. A.

The Amino Acid Sequences of Chains a, b, and c That Form the Trimer Subunit of the Extracellular Hemoglobin from Lumbricus terrestris”

(Received for publication, September 30, 1987)

Kenzo FushitaniS, Maria S. A. Matsuuras, and Austen F. Riggsn From the Department of Zoology, University of Texas, Austin, Texas 78712

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 161, 145, and 163 residues and calculated molecular weights of 17,625, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. BioL Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NHz-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Caz’-binding site in the trimer. Comparison of the sequences of chains u, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Z‘ylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.

The extracellular hemoglobin of the earthworm, Lumbricus terrestris, is one of the most studied of the hemoglobins of annelids (2,3). The molecule has a hexagonal bilayer structure in electron micrographs (4,5). The subunit structure one level lower than that of the whole molecule is apparently a single hexagonal disc and the subunit structure lower than this level

* This work was supported by National Science Foundation Grant DMB-8502857, Welch Foundation Grant F-213, and National Insti- tutes of Health Grant GM28410. A preliminary account of some of this work has been presented (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biochemistry, Dokkyo University School of Medicine, Miba, Tochigi 321-02, Japan.

3 Dept. of Biochemistry, University of Campinas, Caixa Postal 1700, 13100 Campinas, Siio Paolo, Brazil.

ll To whom reprint requests should be addressed.

is a %2 subunit. Although the substructure of the %2 subunit is not clear, the lowest level of subunit structure has been found by dissociation experiments, which show that the molecule is composed primarily of a monomer (chain d ) and a disulfide-linked trimer (chains a, b, and c) (6-9). The nature of additional chains present with size of 31-36 kDa is still uncertain (6, 10). A11 of the four major chains (a-d) have heme and are present in equal proportions (8, 11).

The previously reported amino acid sequence of chain b (as chain AI11 (12) has been found to be partly incorrect. We report here the amino acid sequences of chains a and c and a complete redetermination of chain b.

MATERIALS AND METHODS’

RESULTS

Chain a-Two preparations of chain a gave different products. If the hemoglobin trimer, composed of chains a, 6, and c, is reduced with DTT’ and chromatographed on DEAE- cellulose (8), the major chain a isolated begins with the sequence, His-Ile-Trp-Asp-Asp-, and is designated “cleaved” chain a. If globin is prepared from the trimer and subjected to reverse phase HPLC (Miniprint Section a, Fig. l), “uncleaved” chain a is obtained. The NHz-terminal sequence of this chain shows that cleaved chain a results from proteolytic cleavage of an Arg-His bond at position 16-17 of the chain. Chain a has been sequenced completely by the use of these products. Cleaved chain a was sequenced with peptides de- rived by cleavage with trypsin and Staphylococcus aureus V8 protease. A summary of the sequence determination is given in Miniprint section a, Fig. 2. The complete (uncleaved) chain a has 151 residues and a calculated molecular weight of 17,525 without heme. About 15% of the isolated chain a molecules lack the NH2-terminal alanine and begin with aspartic acid.

Chain b-The amino acid sequence of chain b from L. terrestris has been redetermined. The revised sequence has 145 amino acids residues instead of 157 and its molecular weight is 16,254 without heme. Amino acid residues at positions 55 and 58 were found to be Asp and His instead of His and Asp, respectively. A portion of amino acid residues 123- 157 of the previously reported sequence (12) was revised and assigned to positions 123-145 in the revised sequence as shown in Miniprint section b, Fig. 1. Details of sequence

Portions of this paper (including “Materials and Methods”: section a, Figs. 1-7, Tables I-IV; section b, Figs. 1-6, Tables 1-111; section c, Figs. 4-9, and Tables I-IV) are presented in miniprint a t the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: DTT, dithiothreitol; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; PTH, phenylthiohydantoin; HPLC, high performance liquid chromatography.

6502

Amino Acid Sequences of Chains of Earthworm Hemoglobin 6503

FIG. 1. Comparison of sequences of the four chains, a-d, (13) from Lumbricus terrestris, four chains, IIC, IIA, IIB, and I, from T. heterochaetus (14-17), G. dibranchiuta (18), and the a and @ chain of human hemoglobin (19). Assignment of helices A, B, . . ., H were adopted from human hemoglobin j3 chain (24). F' was adopted from the helical assignments in Glycera globin (25). Solid lines in Glycera (Y and j3 globin sequences were based on x-ray crystallographic data (24, 25). Symbols: * and h, internal and heme contact amino acid residues, respectively, in the j3 chain of human deoxy hemoglobin (26); #, 33 specific positions that are restricted to nonpolar residues (27). See Refs. 24,26, and 28 for roles of specific amino acid residues.

A-2 A-1 I I

A B C D

T y l I T y l I I A T y I I I C T y l 118 L u m a H A H - - - Q R V A F G L E L W K G I L R E H P E I K A P F S R V R G D N I Y - - - - -

L u m a L u m b S G H - - - D R E A F S O A I W R A T F A Q V P E S R S L F RVHGDDTS- - - - -

L u m c

H u m a n a G l y c e r a N D - - - - N G A G V G K D C L I K H L S A H P O M A A Y F G - F S G A S - - - - - - -

H u m a n B """ H A G E Y G A E A L E R M F L S F P T T K T Y , F P H F - D L S H G - - - - - """ N V D E V G G E A L G R L L V V Y P W T O R F F E S F G D L S T P D A V M G

39 4P 5P 60 V G E - - - S R T D F A I D V F N N F F R T N P D - R S L ? & ? N G D N V Y - - - - - V G H - - - E R V E L G I A L W K S M F A O D N D A R D L F K R V H G D V H - - - - - A E D T G - R R T L I G R L L F E E L F E I D G A T K G L F K R V N V D D T H - - - - - A E F T G - R R V A I G O A I F O E L F A L D P N A K G U F Q R V N V D K P S - - - - -

S S F T D - R A V A I V R A V F D D L F K H Y P T S K A L F D T E S S K I K I G F G R L L L T K L A K D I P D V N D L F

a a a x a ax a h * x * I * ** ** h hh hh

I* *

T y l I

T y l I I

T y l I I B T Y I I I C

L u m d L u m b L u m 8

L u m c G l y c e r a H u m a n a H u m a n B

l y l I

T y t I I C T y l I I A

T y l 118 Lum d L u m b L u m a L u m c G l y c e r a

H u m a n E H u m a n a

G H 4 HI1 I I

E F ' F G

N P K V K A H G K K V L G A F S D G L A H L - - - D N L K G T a a a x a * a x a

h h h * X I

* x ** x * x x x x * * * * h h h h h h h h h

H

chain b - C- C- 124 I 1 33

D E V I Y P I K H D

L P A E F T P A V H A S L D K F L - A S V S T V L T S K Y R F G K E F T P P V O A A Y Q K V V - A G V A N A L A N K Y H

a x. a x x a h h h

x * * x *

chain a -C C 6 7

chain c -C c 16 7 139

I C- I

1 I

FIG. 2. Proposed configuration of inter- and intrachain disulfide bonds in the trimer. The numbers C" show the position of cysteine in the individual sequence.

analysis are provided in Miniprint section b. Chain c-The sequence of chain c has been determined.

Chain c has 153 amino acid residues and a calculated molecular weight of 17,289 without heme. Its sequence is aligned in Fig. 1 with those of chains a and b and chain d (13), four chains from Tylorrhynchus heterochaetus (14-17), one chain from Glycera dibranchiuta (18), and human hemoglobin (Y and

/3 chains (19). The proposed arrangement of disulfide bonds is shown in Fig. 2. A phylogenetic tree for annelid globin chains (Fig. 3) has been constructed on the basis of this alignment. The tree is based on analysis of the data in Table I (Miniprint section c ) by an unweighted pair-group method (20, 21).

DISCUSSION

Sequence Alignments-Determination of the amino acid sequence of an extracellular hemoglobin from annelids was first reported for chain b (originally designated chain AIII) from L. terrestris hemoglobin by Garlick and Riggs (12). They reported that they could not place chymotryptic peptide Ch x in the sequence. However, we have found this sequence in chain a at residues 54-66, rather than chain b. We also found that a portion of the COOH-terminal region of the original sequence reported for chain b actually comprised residues 119-127 of chain a (Fig. 1 and Miniprint section b, Fig. 1). These observations showed that the original sequence was in error because of contamination of the preparation of chain b with chain a. Re-examination of the sequence strategy of the original report (12) shows that cleaner peptides and more extensive overlaps should have been utilized. The new sequence reduces the number of residues from 157 to 145 and

6504 Amino Acid Sequences of Chains of Earthworm Hemoglobin Subunlt Arrangement 01 Cysteines

038 7 o M y I I A Trlmer “c-C-C-

b Trlmer - c-c-c-

d Monomer -C-C-

I 050 I Monomer -C-C-

47

FIG. 3. A phylogenetic tree showing relationships and relative evolutionary distances among hemoglobins from L. terrestris (chains a-d), Tylorrhynchus heterochaetus (chains IIC, IIA, IIB, and I), and G. dibranchiata. This tree is based on the sequence alignment in Fig. 1 and the transformed distances in Table I (Miniprint section c ) by using an unweighted pair-group method (20,21). Standard errors at the branching points (half-length of bars), 1-8, are 0.09, 0.08, 0.09, 0.09, 0.09, 0.09, 0.07, 0.14, respectively. Distribution of cysteinyl residues in the extracellular hemoglobin family are shown on the right. Averaged percent identity between two clusters at the corresponding branching points is shown for convenience. Degree of identity between the a and f l chains of human globin is 43.9%.

removes extra residues at the GH corner which had been inserted to get maximal correspondence with the human @ chain (12). The original alignment of the sequence with that of the human @ chain did not show the presence of amino acid residues, Pro(C2) and Phe(CDl), which are highly conserved in other hemoglobins. Although this alignment was adopted with little change by other workers (3,15-17,23), we find that a greatly improved arrangement can be achieved by shifting assignments of the B and C helices for the extracellular hemoglobin 5 residues to the right in the original alignment so that C2(Pro) and CDl(Phe) occupy conserved positions. This shift is compensated by the generation of 3-6 extra residues between the A and B helices, as compared to human a globin, and by loss of the D helix (Fig. 1). The assignment of the G helix by Suzuki and Gotoh (17) has been returned to the earlier one (12) by a 1 residue shift to the right so that Phe(G5) occupies a conserved position. We also removed the gap inserted at position G2 in the original alignment (12) by shifting the last residue of the FG corner in the original alignment to the right so that the new last residue in the FG corner makes a hydrophobic contact with the heme as in other hemoglobins (24). These assignments of a-helices can be rationalized by examining the distribution of internal hydrophobic and heme contact residues as found in the human @ chain and in Glycera hemoglobin. The alignment is also consistent with the distribution of the 33 invariant hydrophobic amino acid residues proposed by Perutz (27, 28) (see legend in Fig. 1). We therefore suggest that the tertiary structures of these globin subunits of hemoglobins from both L. terrestris and T. heterochaetus all have the myoglobin “fold.”

Several characteristics of the extracellular hemoglobin seen from these alignments are as follows. Extra residues appear to be inserted at the AB corner. Such AB insertions are also found in the myoglobin of Busycon (29), and the hemoglobin of Glycera (18,25). The structure of the AB corner with these

additional residues will become apparent with x-ray crystallographic analysis, now underway on the hemoglobin of L. terrestris (30). The present alignment implies that the D helix is absent. The 10 residue segment between the C and E helices may form a non-helical corner as in the Glycera (25) and human a (24) chains which have 7 and 9 residues, respectively, for this segment. Since the number of amino acid residues between the E and F helices are the same as in Glycera hemoglobin, the secondary structure in the EF corner may be similar to that of Glycera hemoglobin in forming an F’ helix (25). The GH corner and H helix have been aligned as in the Glycera and human @ chains. This alignment shortens the H helix by 3 residues and can be rationalized by the presence of a bulky hydrophobic residue at GH5 which plays the role of spacer between the G and H helices and contributes to the hydrophobic cluster at the bottom of the heme pocket (24,28) and by the presence of H8 Trp which is also found in Glycera hemoglobin. The position of the COOH-terminal end of the H helix seems uncertain.

We rearranged a portion of the FG corner to G helix region of chain I in T. heterochaetus hemoglobin (17) to the right 3 residues, accompanied by a deletion at the FG corner and an insertion of 3 residues at the GH corner. This shift (see Fig. 1) appears to be consistent with other sequence alignments and the distributions of hydrophobic residues in the FG corner to the G helix, such as residues at FG7, G5, G8, G12, and G16. This alignment indicates that deletions or insertions occur almost entirely in interhelical segments or at the NH2 or COOH termini (31). One apparent exception is an insertion between H12 and H13 in T. heterochaetus chains I and IIA. This insertion was positioned to get maximal correspondence among the globin sequences, but such a change in the middle of a helix is likely to cause severe problems in forming the appropriate folding.

Suzuki et al. (14, 15) reported no conserved residues in the COOH-terminal region after (G5) Phe. However, the new alignment suggests no significant differences in extent of correspondence in this region compared to the region from the A helix to the CD corner. We conclude that their obser- vation resulted mainly from use of the earlier sequence of chain b which has now been corrected. One significant differ- ence was observed between chains I and IIA, and I and IIB in T. heterochaetus. The phylogenetic tree (Fig. 3) suggests that chains I and IIA belong to the same subfamily. The G helix of chain I appears anomalous, however, and shows little correspondence to any other G helix except that of chain IIC which is in the other subfamily.

The most striking finding from the sequence alignment is that all positions of the 10 cysteinyl residues in the chains of both L. terrestris and T. heterochaetus are conserved in the homologous chains (Fig. 1). It is particularly noteworthy that the A-l3 and HI1 cysteinyl residues are all conserved. The corresponding positions in vertebrate hemoglobins (NA2 in a chain or NA3 in @ chain and H11) have conserved hydrophobic residues that make internal contacts (24, 26, 28). These cysteinyl residues therefore appear to be close enough to form an intrachain disulfide bond in the tertiary structure. The position of this bond in chain d has been chemically determined and no free -SH groups in the trimer have been found: Therefore all cysteinyl residues in the trimer (chains a, b, and c ) must participate in intra- or interdisulfide bonds. We assume that each chain in the trimer has the same intrachain disulfide bond as that determined for chain d. If SO, the

3A-1 designates 1 residue, A-2 designates 2 residues, before the start of the A helix.

K. Fushitani and A. F. Riggs, unpublished observations.


remaining 4 cysteinyl residues, two at A - P in chains a and c, and the other two at GH4 in chains a and b, must participate in interchain disulfide bonds to form the trimer. Experiments4 have shown one of the two possible interchain disulfide con- figurations; one of the interchain disulfide bonds is between the cysteine at A-2 in chains a and c, and the other is between the cysteine at GH4 in chains a and b (Fig. 2). This suggests that the positions of both intra- and intersubunit disulfide bonds and the resulting arrangement of subunits in both the trimer and the 1/12 subunit are identical in the hemoglobins of L. terrestris and T. heterochaetus. This is consistent with the observed similarities in shape and size of the extracellular hemoglobins of the Oligochaeta and Polychaeta (23).

Heterogeneity-Heterogeneity was found in both chains a and b. Two tryptic peptides from chain b, T7 and T7’, were found in the proportion of about 2:l. The amino acid composition (Miniprint section b, Table I) and sequencing data (Miniprint section b, Table 11) showed that they were identical except that T7 had Val and T7’ had Ala at position 41. We found no other peptides showing heterogeneity in the chro- matogram of the tryptic peptides. This suggests that heterogeneity in chain b is restricted to a very small number of positions. In contrast, the heterogeneity reported for chain d (13) seems to occur in both NH2- and COOH-terminal segments. These facts indicate some uncertainty concerning homogeneity with respect to chains b and d. The heterogeneity might be present in individual molecules to produce hybrids or it might represent a polymorphism. A large number of worms (>1000) were used in preparing the material so that this question cannot yet be answered.

Sequence heterogeneity was observed at the NH, terminus of chain a. The major chain (82-86%) starts with Ala-Asp- Asp- and minor chain (14-18%) starts Asp-Asp- (see peptides T1 and T1-2 in Tables I and 11, Miniprint section a). The origin of this heterogeneity is uncertain but it might arise either from the action of a signal endopeptidase or from the presence of two genes. It seems possible that the signal peptidase might cleave at two sites: 100% at the first site and 14-18% at the second site. Cleavage of an Ala-Asp bond is consistent with the known specificity of signal endopeptidase which cleaves after small residues such as Ala, Gly, or Cys (32, 33). This is also consistent with the finding in our laboratory that the cleavage of the signal peptide from chain c occurs at an Ala-Asp bond (65). Cleavage also occurs after Ala in the signal peptide from Chironomus globin (34). It is not possible to test for the presence of chain a lacking Ala and starting with Asp within the trimer because chain c also begins with Asp.

The Arg-His bond at position 16-17 appears to be labile. Reduction of the trimer (as hemoglobin) followed by chromatography on DEAE-cellulose results in 100% cleavage of this bond. This cleavage does not occur in reduced and carboxymethylated globin prepared from the trimer and subjected to HPLC. The proteolysis seems unlikely to have resulted from adventitious contamination with a protease but we cannot completely exclude this possibility. The cleavage is reproducible and quantitative in different preparations.

Gotoh et al. (11) recently reported the NH,-terminal sequence of the first 22 residues of chain a and identified Glu, Ser, and Val a t positions 3, 5, and 15, respectively. We obtained the same identifications at positions 3 and 5 in our initial sequence analysis of the intact chain a before we were aware of the heterogeneity at the NH, terminus. However, the amino acid composition and sequence data (Miniprint section a, Tables I and 11), clearly show Asp at both positions 3 and 5. Since part of degraded carboxymethylated PTH-

cysteine elutes at the position of PTH-Ser in our chromatography system, a misidentification could result. The amino acid residue at position 15 is Ile instead of Val in our sequence. This is obvious from amino acid composition data (peptide T2 in Miniprint section a, Table I), although Val might have been expected from the sequence comparisons (11). We conclude that the differences observed between the two sequences result from technical problems and do not reflect intrinsic differences.

Calcium-binding Site-Oxygen equilibrium measurements of the trimer in the presence and the absence of Ca2+ have shown that the trimer has at least one oxygenation-linked Ca2+-binding site (8). The proposed interchain disulfide bond between the cysteinyl residues in position 6 of chains a and c could put the NH2-terminal sequences of chains a (ADDEDC-) and c (DEHEHC-) close together (Fig. 2). This structure would contain a total of seven negatively charged carboxyl groups and two histidines. This cluster seems likely to constitute at least part of a primary calcium-binding site. If one Ca2+ ion is bound per trimer and four trimers occur in each 1/12 subunit, 48 Ca2+ ions would be bound per intact molecule, a value consistent with those reported for the hemoglobins of the related annelids, Octahium (35) and Tubifex (36). Oxygen equilibrium measurements of the whole molecule indicate that 0.77 protons are released and 0.37 Ca2+ ions taken up for each 0, bound at pH 7.4, or 2 protons/Ca2+ ion (37). We suggest that the imidazole groups of the histidyl residues in positions 3 and 5 of chain c might form electro- static links with the aspartyl groups at the same positions in chain a in the deoxy hemoglobin. Oxygenation would then be accompanied by a structural perturbation such that the his- tidy1 residues would move away, thus lowering the pK values. Since the Bohr effect is zero in the absence of calcium, proton release and calcium binding must be tightly linked (37, 38). The corresponding chains of Tylorrhynchus hemoglobin lack the ADDEDC and DEHEHC extensions before the A helix; the corresponding chains have only DTC- and DDC- respectively. Unfortunately, no data are available on the effect of calcium on oxygen binding by this hemoglobin.

Phylogeny-A correspondence matrix (Table I, Miniprint section c) was made from the alignment in Fig. 1. We have constructed a phylogenetic tree (Fig. 3) from the data of Table I. The standard errors are given at the branch points (20,21). The pattern suggests the following: 1) a duplication of the ancestral gene occurred to produce two lines, one coding for intracellular hemoglobin and the other becoming a gene for extracellular hemoglobin. The latter gene must have acquired DNA coding for a signal peptide to make possible secretion of the hemoglobin. Acquisition of a cysteinyl residue at position A-1 and H11, and deletion of the D helix and the beginning of the H helix must also have followed this duplication. 2) The gene for the ancestral extracellular hemoglobin with a signal peptide duplicated at least twice to produce four chains. The first duplication in the extracellular hemoglobin gene line produced two subfamilies, ac and bd. The sequence of the relevant genomic DNA should provide the information required to determine whether two ancestral subfamily genes duplicated independently or as a set. Deletions or insertions at the NH,-terminal region, the AB and FG corners, and at GH3 must have occurred after the first gene duplications in the extracellular globin gene line. Acquisition or loss of the cysteinyl residue at A-2 also must have occurred in the ac or bd subfamily, respectively. A lysyl residue at AB7 in chain c seems to have been inserted after separation of Oligochaeta and Polychaeta. The distribution of cysteinyl residues at GH4 within and between subfamilies is not consistent with this

6506 Amino Acid Sequences of Chains of Earthworm Hemoglobin

picture because we would expect that cysteinyl residues at GH4 would be localized in either the ac or bd subfamily. However, their distribution is across both families. This prob- lem might be explained by assuming that an independent mutation occurred at GH4 or that an exchange of a portion of a gene took place after the genes for the four chains were produced. Duplication events and distribution of cysteinyl residues are all the same in L. terrestris and T. heterochaetus. This indicates that these gene duplications preceded the separation of the Oligochaeta and Polychaeta.

It is well known that amino acid substitution rates in positions with structurally and functionally important roles are lower than those elsewhere (39, 40). Extracellular hemoglobins must have different structural constraints compared to those of intracellular tetrameric hemoglobins of vertebrates. If one assumes nonetheless comparable evolutionary rates for extracellular and vertebrate hemoglobins, divergence of the Oligochaeta and Polychaeta would have occurred around the time of the gene duplication which led to the cy and @ gene families in vertebrates.

Annelids show considerable variation in types of hemoglobins. Thus, GZyceru has only intracellular hemoglobin (41), L. terrestris, and T. heterochuetus have only extracellular hemoglobin, but Amphitrite ormta (41) and Travisia japonica (42) have both types of hemoglobin in individual worms. Fushitani et al. (43) showed that antisera made against the extracellular hemoglobin of T. japonica did not react with its intracellular hemoglobin on the basis of immunodiffusion tests. This fact is consistent with the phylogenetic relation- ship shown in Fig. 3 that indicates that intracellular and extracellular hemoglobin diverged very early. The finding is also consistent with the report that sequence changes on the surface of the molecule which exceed 40% are usually suffi- cient to eliminate immunological cross-reactivity between two proteins (44). The values in Miniprint section c Table I show that the extracellular hemoglobins are equidistant from the intracellular human cy and @ chains and Glyceru globin. This suggests that the lines leading to Glycera hemoglobin and the ancestors of the (Y and @ globins of vertebrates diverged after the intra-extra cellular split.

Organisms can be classified into five kingdoms (one pro- karyotic and four eukaryotic) (45) in all of which hemoglobin has been found (24, 46-48). Several nonvertebrate hemoglobins occur that are quite different in protein structure and in gene structure from those of vertebrate hemoglobins (34,49- 52). Recent x-ray crystallographic studies of a molluscan hemoglobin (53) have clearly demonstrated that even the small intracellular tetrameric hemoglobins are assembled dif- ferently from vertebrate hemoglobin. The sequence of cDNA for globin from the red cells of the clam, Barbutiu, has been shown to code for a polypeptide chain that contains two myoglobin-like domains (52). One classification of hemoglobins is based on structure and assembly (3). We suggest the following criteria for classifying globins: (i) single or multi- domain with the number of domains based on the number of myoglobin “fold” units; (ii) intracellular or extracellular hemoglobin (these will differ at the biosynthesis level by the presence or absence of a signal peptide; (iii) chaemeric type or not (fused genes of different types of protein as suggested for yeast hemoglobin (48)); (iv) myoglobin type or hemoglobin type.

Nomenclature-Barcroft and Barcroft (54) used the term “hemoglobin” for invertebrate respiratory proteins on the basis of studies of absorption spectra and of the relative affinities of oxveen and carbon monoxide. Although Svedberg

respiratory proteins and vertebrate hemoglobins, they claimed that “this does not necessarily mean that mass and the chemical properties of the protein part of the molecule are the same as those of the haemoglobin of the vertebrates” based on the finding of high molecular weight by ultracentrif- ugation and low isoelectric point. Accordingly, they revived the term “erythrocruorin” for invertebrate hemoglobins. The fact that these high molecular weight hemoglobins were found in plasma instead of cells appeared to provide strong evidence to the idea that erythrocruorin was unique. Later, Levin (4) concluded that there were probably no structural similarities between hemoglobin and erythrocruorin on the basis of studies by electron microscopy. Recent studies (12, 13, 14-17 and the present work) have shown that the criteria on which Svedberg and Eriksson and Levin based their proposals for the term erythrocruorin are no longer valid. Variation in size, isoelectric point, and degree of aggregation of invertebrate hemoglobins and existence of hemoglobin in other kingdoms make a definition of erythrocruorin completely obscure. Since erythrocruorin proteins are clearly members of the same globin family, naming them all hemoglobins would be advan- tageous. We think usage of erythrocruorin should be abolished as previously proposed (3, 12,57).

The naming of isolated polypeptide chains from extracellular hemoglobins of annelids on the basis of isoelectric point, amino acid analysis, or band pattern on SDS polyacrylamide gel electrophoresis is clearly arbitrary. It would be advanta- geous to have a common nomenclature for a pair of homologous polypeptides within the same kind of hemoglobin from different organisms. On the assumption that constituent polypeptide chains of the extracellular hemoglobins of annelids do correspond, four of their chains can be named according to our present terminology (Fig. 1). We propose the following experiments to serve as criteria for classification of chain type: (i) gel filtration in the absence of reducing reagent (chain demerges as a monomer); (ii) blocking possible free sulfhydryl groups in the trimer, followed by reduction of -S-S- bonds and isolation of three chains, and sequencing the first 10 residues of each chain (chain b has one cysteinyl residue in this region); (iii) titrating cysteinyl residues of the remaining two chains (chain c has three and chain a has four cysteinyl residues).

Acknowkdgrnents-We wish to acknowledge the expert assistance of Sandra Smith, the University of Texas Protein Sequencing Center. We also thank Karen Haschke for assistance. We thank Patricia Q. Behrens for helpful discussions. We thank Takashi Takagi, Univer- sity of Tohoku, Sendai, Japan and Tomohiko Suzuki, University of Kochi, Kochi, Japan for suggestions for the technique of the manual Edman degradation. We thank the University of Southern California Comprehensive Cancer Center for determining the sequence of some peptides with a gas phase sequencer. We thank Dr. Masatoshi Nei, University of Texas at Houston, for help in the construction of the phylogenetic tree and for valuable discussions.

REFERENCES 1. Fushitani, K., and Riggs, A. F. (1987) Biophys. J. 51,301 (abstr.) 2. Vinogradov, S. N., Shlom, J. M., Kapp, 0. H., and Frossard, P.

3. Vinogradov, S. N. (1985) Comp. Biochem. Physiol. B Comp.

4. Levin, 0. (1963) J. Mol. Bwl. 6, 95-101 5. Kapp, 0. H., Vinogradov, S. N., Ohtsuki, M., and Crewe, A. V.

6. Shlom, J. M., and Vinogradov, S. N. (1973) J. Bwl. Chem. 248 , 7904-7912

7. Vinogradov, S. N., Shlom, J. M., Hall, B. C., Kapp, 0. H., and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155

(1980) Comp. Biochem. Physiol. B Comp. Biochem. 67, 1-16

Biochem. 82 , l -15

(1982) Biochim. Biophy~. Acta 704,546-548

and Eriksson (55) saw the resemblance between invertebrate 8. Fushitani, K., Imai, K., and Riggs, A. F. (1986) in Invertebrate “I


Oxygen Carriers (Linzen, B., ed.), pp. 77-79, Springer-Verlag, 33. Davis, B. D., and P.-C. Tai (1980) Nature 283,433-438 Berlin 34. Antoine, M., and Niessing, J. (1984) Nature 310 , 795-798

9. Shishikura, F., Mainwaring, M. G., Yurewicz, E. C., Lightbody, 35. Chiancone, E., Bull, T. E., Norne, J.-E., Forsen, S., and Antonini, J. J., Walz, D. A., and Vinogradov, S. N. (1986) Biochim. E. (1976) J. Mol. Biol. 107 , 25-34 Biophys. Acta 869,314-321 36. Rokosz, M. J., and Vinogradov, S. N. (1982) Biochim. Biophys.

10. Vinogradov, S . N., Lugo, S. D., Mainwaring, M. G., Kapp, 0. H., Acta 707, 291-293 and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 37. Fushitani, K., Imai, K., and Riggs, A. F. (1986) J. Biol. Chem. 8034-8038 261,8414-8423

11. Gotoh, T., Shishikura, F., Snow, J. W., Ereifej, K. I., Vinogradov, 38. Santucci, R., Chiancone, E., and Giardina, B. (1984) J. Mol. Biot. S. N., and Walz, D. A. (1987) Biochem. J. 2 4 1 , 441-445 179,713-727

12. Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257,9005- 39. Dickerson, R. E. (1971) J. Mol. Evol. 1, 26-45 9015 40. Kimura, M. (1983) in The Neutral Theory of Molecular Evolution,

13. Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and pp. 98-116, Cambridge University Press, Cambridge Walz, D. A. (1987) J. Biol. Chem. 2 6 2 , 3123-3131 41. Terwilliger, N. B., Terwilliger, R. C., and Schabtach, E. (1985) in

14. Suzuki, T., Takagi, T., and Gotoh, T. (1982) Biochim. Biophys. Blood Cells of Marine Invertebrates (Cohen, W. D., ed) pp. 193-

15. Suzuki, T., Yasunaga, H., Furukohri, T., Nakamura, K., and 42. Ochi, 0. (1969) Mem. Ehime Uniu. Sci. Ser. B (Biol.) 6 , 23-91

16. Suzuki, T., Furukohri, T., and Gotoh, T. (1985) J. Biol. Chem. Physiol. B Comp. Biochem. 7 2 , 267-273

17. Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 2 6 1 , 9257-9267 7010-7017 18. Imamura, T., Baldwin, T. O., and Riggs, A. S. (1972) J. Biol. 45. Whittaker, R. H. (1969) Science 163 , 150-160

Acta 708,253-258 225, Alan R. Liss, Inc., New York

Gotoh, T. (1985) J. Biol. Chem. 2 6 0 , 11481-11487 43. Fushitani, K., Ochi, O., and Morimoto, H. (1982) Comp. Biochem.

260,3145-3154 44. Prager, E. M., and Wilson, A. C. (1971) J. Biol. Chem. 246,

Chem. 247,2785-2797 46. Wakabavashi, S., Matsubara. H.. and Webster. D. A. (1986) 19.

20.

21.

22. 23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Hunt, L. T., Hurst-Calderone, S., and Dayhoff, M. 0. (1978) in Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed) Vol. 5, Supplement 3, pp. 229-264, National Biomedical Re- search Foundation, Washington, D. C.

Nei, M. (1987) in Molecular Evolutionary Genetics, pp. 287-326, Columbia University Press, New York

Nei, M., Stephens, J. C., and Saitou, N. (1985) Mol. Biol. Evol.

Deleted in proof Vinogradov, S. N. (1985) in Respiratory Pigments in Animals

(Lamy, J., Truchot, J.-P., and Gilles, R., ed) pp. 9-20, Springer- Verlag, Berlin

Dickerson, R. E., and Geis, I. (1983) in Hemoglobin, pp. 37, 68- 69, The Benjamin/Cummings Publishing Co., Menlo Park

Padlan, E. A., and Love, W. E. (1974) J. Bid. Chem. 249,4067- 4078

Sack, J. S., Andrews, L. C., Magnus, K. A., Hanson, J. C., Rubin, J., and Love, W. E. (1978) Hemoglobin 2 , 153-169

Perutz, M F., Kendrew, J. C., and Watson, H. C. (1965) J. Mol.

Fermi, G. and Perutz, M. F. (1981) in Haemoglobin and Myoglobin, Atlas of Molecular Structures in Biology (Phillips, D. C., and Richards, F. M., ed) Vol. 2, p. 102, Clarendon Press, Oxford

Thompson, E. 0. P. (1980) in The Evolution of Protein Structure and Function, UCLA Forum in Medical Sciences 21, pp. 267- 298, Academic Press, New York

Royer, W. E., Jr., Hendrickson, W. A., and Love, W. E. (1987) J. Mol. Biol. 1 9 7 , 149-153

Bajaj, M., and Blundell, T. (1984) Annu. Reu. Biophys. Bioeng.

Strauss, H. W., Zimimerman, M., Boime, I., Ashe, B., Mumford, R. A., and Alberts, A. W. (1979) Proc. Natl. Acad. Sci. U. S. A.

2,66-85

BWl. 13,669-678

13,453-492

76,4225-4229

, , . , Nature 322,481-483

47. Usuki, I., and Irie, T. (1983) Comp. Biochem. Physiol. B Comp. Biochem. 75,421-424

48. Oshino, R., Asakura, T., Tamura, M., Oshino, N., and Chance, B. (1972) Biochem. Biophys. Res. Commun. 46 , 1055-1060

49. Terwilliger, R. C., Terwilliger, N. B., Bonaventura, C., and Bon- aventura, J. (1977) Biochim. Biophys. Acta 494,416-425

50. Moens, L., Van Hauwaert, M. L., Geelen, D., Verpooten, G., and Van Beeumen, J . (1986) in Invertebrate Oxygen Carriers (Lin- Zen, B., ed) pp. 81-84, Springer-Verlag, Berlin

51. Deleted in proof 52. Riggs, A. F., Riggs, C. K., Lin, R.-J., and Domdey, H. (1986) in

Invertebrate Oxygen Carriers (Linzen, B., ed) pp. 473-476, Springer-Verlag, Berlin

53. Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316 , 277-280

54. Barcroft, J., and Barcroft, H. (1924) Proc. R. SOC. Lo&. B Biol. Sei. 9 6 , 28-42

55. Svedberg, T., and Eriksson, I.-B. (1932) Nature 130,434-435 56. Deleted in proof 57. Keilin, D., and Hartree, E. F. (1951) Nature 168, 266-269 58. Fushitani, K., Morimoto, H., and Ochi, 0. (1982) Arch. Biochem.

59. Fushitani, K., Bonaventura, J., and Bonaventura, C. (1986) Comp. Biochem. Physiol. B Comp. Biochem. 8 4 , 137-141

60. Teale, F. W. J. (1959) Biochim. Biophys. Acta 35 , 543 61. Tarr, G. E. (1977) Methods Enzymol. 47,335-357 62. Deleted in proof 63. Tarr, G. E. (1981) Anal. Biochem. 111, 27-32 64. Mahoney, W. C., Smith, P. K., and Hermodson, M. A. (1981)

65. Jhiang, S. M., Garey, J. R., and Riggs, A. F. (1988) Science, in

Biophys. 218,540-547

Biochemistry 20,443-448

press.


I .o

0.8

E 5 0.6 N 0 c

0.2

c

m i c acid

80

70

60

50

40

30

20

irj w,'-qlo 1 0 20 30 40

Time(min1

__i k -I - v3-3 v4 Y5-3 -

v3-2

v5-5

I " I I v 3 V5-I v5-2

v5


I .c

0.6

s 2 $j 0.6

W 0

0 c 0

fl 0.4 2

C l

U

0.2

C

1 - 1

140

I

'0 1 s 10 20 30 40 -0

Time (min) Time(min)

TI TI-1 TI-2 T2 T3 T4 TS T6 T7 7 8 T8-I T8-2 T9 TI0 TI1 TII-1 Tll.2 TI2 TI3 TI4

Told 13 I2 12 3 15 5 8 6 Pauian 1-13 1-12 2-13 14-16 11.31 32-36 37-48 45-50 53-55 56-61 5657 58-67 68-12 73.104 105-1w 105 1(M109 11a-113 114-121 122-137 Posilion Yield(%) 8 3 3 25 27 36 45 33 37 28 I7 16 42 34 9 9 41 48 32 Yicldl'b)

5 I2 2 1 0 5 3 2 5 1 4 4 8 16 Total

Table 1. (conlinued) Chain a: Amino acid composition of w ! i c gnd pmtcax V8 peptides. Valuer io pmntheexr M number of rcriducr determined by wquencing.

TI5 TI6 TI7 TI8 VI VI-] VZ V3 V3-I V3-2 V3-3 V4 V5 VS-l VS-2 V5-3 V5.4 VS-5

6.5(6) 2.2(2) 2.3(2) 4.1(4) 3.1(3) 2.4(2)

0.2

].I(]) 1.211) 1.0(1) 1.0(11 0.9(11 0.9(1)

1.0111 0 2

2.0(2) 1.2(1) 1 K 1 ) 1.1(11 1 . 1 m 2.0(2) 02 0.2

1.1(2) 0511) 0.6(1) - (1) 4.60) 2.1(2) 2.9(31 2.0(21 2.1(21 2.9(31

3.7(4] 2.813) I.0(1) 2.0i2) I.O(I) 0.8111

0.2

TOW 4 4 3 3 36 14 8 45 21 19 I 1 Porinon 138-I41 102-145 146648 149-151 11.54 41.54 55-62 66-110 66-86 92.110 lODll0 111-117 118-151 118-133 134.151 118-126 139-151 134-149 POSitiOn YicldlB) 37 39 49 31 5 3 27 3 5 7 3 18 5 2 3 2 1 0.1 Yleld(B1

1 34 16 I8 I I 13 16 Total


2 (1) I IH)

3 w 4 D 5 D 6 V 7 w 8 (SI 9 (SI 10

:: 5) 14 R 15 @) 16

18 I 17 A

19 V ;y '9 22 v 23 F 2 4 D 25 D

27 F 26 L

28 K 29 H 30 Y 31 P

33 $1 32 m 34 35 A 36 37 38 E 39 (R) 40 V 41 K 42 I 43 D 4 4 E 45 P 46 E

14.7

D 7.9

v 7.5

I 2 3

26 5 23 6 30.7 18.1 10.4 13.5 8.6 6.7

6.1 13.5 13.4 10.4 4.4

d

F 6.7

D 4.8 R 1.7

v 9.2

v 7.4 10.8

1 2 3

H 9 4

6.0 8.5

3.2 4.9

9.0

7.0 8.2

Y P T F K

4 5 6

2.7 1.7

15.4 8.0

15.7 9.1

16.0

15.8 I3 2

A L F E R

A 5.7 V 7.0

D 3.8 F 1.3 13.9

9.0

7.6 6.7 8.7

6.3 3.1

2.0 3.1 4.4

V K I D E P E s G E F K s H V R

V A N G L D L L I

19.8 19.4 15.9 6.4

11.2 4.0

16.3 IO 9.2 7.5 7 6 1 3

L 3.6

K 0.9 F 3.3

H 2.2

P uacc Y 3.0

6

8 9 10 11 12 1.7

5.0

4.8 3.1

A 4.9

13.4

7.2 5 2

6 4 2.5

E2.1

V 1.3 K 1.4 5.5

0.7 2.1 2.2 2.6 0.9 2.8

37.8 69.5 31 6 25.4 36.2 16 3 28 5 26.9 22.5 24.5 24 0 25.8

0.5

2 1 I H

3 w 4 D 5 D 6 V

21.6 14.3

42.4 15.3

17.3 16.4

w mce 16.2

D 11.2 8

io 12 13

N

L D D T L V L

8 L G H L A D

9 P

w wcc v 7.9

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 M 31 32

6.4 2 7

21 I 16.5 I8 4 10.3 1.9

2 D 1 A

3 D 4 E S D 6 (C) 7 (C) 8 (:)

11 D 10 E

I2 R 13 R 14 E I5 I 16 R 17 H 18 I 19 w

22 9 D 5.0

E 7.2 D 7.2

~~. 9.7

13.3 17.3 8.4

22.0 9.0 5.2

14.0 14.5 4 2 5.2 2.9 5.9

i o Y 5.8

D 2.6 R 3.9

12.5 10.3

122 6.2

9.9 6.0

12.4

3.3 6.0

7.7 2.9

E 6.0 17.5 R 4.7 H 2.7 164 4.1

I .7 D 2.5

A 0.4 A 0.2 D 1.3 E0.6

A 0.4

E Y F R

2 7 1.6 2.5 3.3

1 2 3 4

2 D I A

3 D 4 E 5 D 6 C

1.3 0.7 0.8 0.8

UaFC 1.1 I

2 3

r; 15 3 25.3 15 0 18.5 22.7 16.9 14.6

8 2

c A F

2 D I D

3 E 4 D 5 c

7 s 6 C

B Y 9 E 10 D 11 R

2.4 2.1 1.0 1.1

w c c UaCC

0.2 0.5 0.5 0.4 0.2

5

7 8

D 0.8

D 0.8 F 0.4

D 0.3 D0.2

Y 0.2 s 0.1

D 0.2 E 0.2

A

V L P s s C F N V D A W N R

D L P

F K s H L V R V A N

TI4

wqucncn (35)

22 0 M.8 8 2

16.6 20.2 18.0 2.0

11.9 12.8

19 3 11.3

6 3 18.5 7.3 6 6 5.7

1 E 2 1 3 R

5.3 2.5 7.1

E 1.0 8

10 11 12 13 14 I5 16

2 1 1 H

3 w 4 D 5 D 6 V 7 w 8 s

10 s 9 s

11 F I2 T 13 D 14 R IS R

I V 2 A

4 v 3 1

5 R

13.8 13.8 7.7 9.4

14.7 6.9

6 5 1.9

13.4 1.2 2.0

mce

w 1.1 I 4.5

D 1.)

w trace V 2.3

S 0.8 1 2 3

0.7 P0.2 8 4

0.3 T uace F 3.9

D 1.7 R 2.1 D 1.1

1 2 3 4

20.1 18.1 3.3 9.5

22.5 18.6

15.4 7. I

19.6 3.4

6. I 9.5 5 4

5 6 7 8 9 10

T4

manual (14.2)

4.7

3.4 5.7

7.2 3.7


v3-3 I 1301 2 quencer 3

4 5 6

2 15.4 11.8

P 11.7 11.7 5.5

12.5 G 7.3 V 9.5 T I .7 K 6.1 E 4.0

8

10 11

v 4 1 1401 2 rcqucncer 3

4 5

Y F 32.3

30.6

R 33.2 G 22.3 I 22.3 0 12.9 E 5.0

ryucnccl. The original idcnrificarion of His at position 55 depended on manual rcqusncing of peptide ChSa (12). Position 55 had bcen idcnrified in ELCD 7 of ChSa sohlv on the bans of a m ~ e r WE! which

Two P M S of the q v s n c s required comction: in positions 55-58 and 123-157 (numbering of mgmai

6 7

v 5 - l I (201 2 rcqequenccr 3

4 5 6 7 8

A 17.1 F 16.6 A 20.9 R 15.8 V 12.0 L 12.3 P 2.5 Q 8.3

A W

36.6 13.2

N 12.3 R 8.2 C 5.0 F 11.3 H R 17.9

9.6

L 11.1 V 12.4 A 11.4 R 7.5 I 4.3 A 16.4 K 5.1 D 2.8 L P mace

2.7

8 9 10 11 12 13 I 5 14

16 17 18

Table 111. Amino acid canpontion of chain a.

Analysis of by q u e n c c Analyns rcriducr

1-16 17-151 clcaveda Canp1cte f sequence

Amino Actd

19.9 15.5

4.6

13.4 9.0

3.8

10.5 7.1 1 8

3 ;>lo 4923 4 4.4

6. I 11.2 1.9 12.9

0 7.9 16.2 3.1 8.7 7.9 6.9

14.1

4.5 6.2 10.2

12.9 0

6.9 16.5

9.0 1.9

7.2 6.5

10.6

d 6

11 4 13 0 8

16 3 9

1 10 2 2

13 0

I 7 16

I 2

6

0 7 7 3

3 11

i 14

v5 T3 T3* T3' T3'* T3X

1.0

1.0 1.0 1.0 1.0 1.1

0.9 10 0.9 0.9 1.0

1.1 1 1 1.1 1.0 1.1

1.1 1.1 1.0

2.2 2.2 1.1 1.2 2.2

Ilc Le"

2


1.0,

5 N % 0.6-

rn u 0 e 0.4 - u) D Q

0.2 -

OO b

Timeh in )

3, 5

TI I

12'

/

4

I .c

0.8

E 2 0.E cu 0 0) 0

0

L

n 0.4 Is a

0.2

OO- Time (rnin)

Fig.6 Reehrormtogaphy of -tis peplids fraction (TI0 i TI 1-2) obmined PI shown in Fig.3. CDndltionl wme PI in Fig5 Gradienr; 18-23 % for 30 min.

Table 1. Chab b Amino Acid Comporition of p m l ~ r e V8 and -tic peptide%. Values in p a ~ l h c r r am the nvmbsrof residues determined by qusncing.

VI V2 V3 V3-1 V3-2 V4 V5 V5-I V5-2 VS-3 V6 V6-I Vl W - I V8 V8-I V8-2 V8-3 V8-4 V9 VI0 TI

T d 8 1 I2 6 6 16 30 23 18 14 22 I5 9 1 31 9 21 8 13 10 1 1 Tom

Yield(%) 22 21 2 14 12 I8 0.3 19 3 0.3 12 14 35 3 I5 14 5 5 8 19 21 10 Yield(%) PolitiOn 1-8 9-15 1627 1621 22-27 28.43 44-13 44.66 49-66 53.66 61-88 14-88 89-97 91.97 98-128 98.106 108-128 108-115 116128 129-138 139-145 1.2.IW Position

Amino Acid Sequences of Chains of Earthworm Hemoglobin Table I. (continued) Chain b Amino Acid Composition of protcaw V8 and myptic peptides. Values in paxnhsar ax the n u m k of residues determined by ryucncing.

R T3 T4 TS T6 T7 T7' T8 'l9 TI0 TI1 TI1-l T11-2 TI2 T12' TI3 TI4 TIS TI6

6513

1.0(1) 0.2

1.0(1) 0.9(1) 2.1(2)

1.0(1) 2.1(2) 2.0(2)

2.2(2)

2.0(2)

0.7W 1.0(1) I.WL)

2.1(2)

1.0(1) 1.ciL)

0.4

0.2

1.0(1)

1.0(1)

1.0(1)

T O I ~ 9 2 s 8 9 lo I O 4 I 17 32 19 13 I I IO 13 4 IS 3 tal Position 3-11 12-13 14-18 19-26 27-35 36-45 3645 46-49 SO 51-67 68-99 68-86 87-99 IW-110 101-110 Ill-123 124-127 128-142 143-145Posltion Ylcld(%) 9 8 17 19 16 13 I 7 6 I 1 8 5 4 5 6 9 12 8 10 Yield(%)

Table 11. Chain b Scqucncc analysis of peptides obramed by cleavage with mypr~n and U&lw&cu V8 protease.

Peptide Y d d Background (nmolcs) Cycle Amino Aad (nmolcs) (nmoler)

Table 111. Amino dad comporilion ofchain b

Ammo Acid by sequence by analysi~

T7 I A (10)

7.8 2 T

Manual 3 7.9

F Edman 4 A 0.3

0.4

2 9 0.2 0.3 € 0 2

0.1 8 E 0. I 9 S 0. 1

10 ( R )

I 2 3 4

1.8 1 . 3 1.4 I .7 0.9 E 0 2 1.1 1.1

0.3 0.8

(3.7) Manual Edmsn

5 6

i In 9

Total 145 V H G D D T S H P A F I A H A E R

21 2 11.1 13.5 10.5 12.5

11.7 1.6

31.0 7.3

16.9

22.0 16 2

12.1

12.7

0.8

16 n 1 2 0

8 9

1 0 11 12 13 14 I S 16 17

TIS

sequencer (45)

E A w

25 5 10.4

22 I 31.5 21.8 mce 29.6 16.8

12.9 23.8 15.1 10.7 4.1 3.6

27.0

L 2 3 4 5 6 7

9 10 I I I2 I3 14 IS

n

D A C I D H I E D G I K

V V 9.2

8.1

12.5

5.3 8.7 2.6 6 0

macc 7. I 2.4

1.4

9.0

9.0

8 9

I0 11 12 13

VI0 manual

I 2 3 4

24.4 11.2 9.9

0.5 0.5

0.9

0.8

5 6 7


1.07

0.8 -

E 0.6-

cu

a 0

0 c 0

c

0.4- 13 In a

0.2 -

O f

TRPAZ

RPA3

TRPA4

1 7o 60

50

40 8 ID

30

20

I O

I I I I I \

10 20 30 40 50 Time ( m i d

I .c

0.8

E 2 0 .E cu 0 a u c 0

c

g 0.4

a A3 v)

0.2

C Time(min)

Fig.8 Rcchmmalogm hy of peptide frrtion T14 + T19 isolavd as shorn in F~g.6, on HPLC. The suns column was u d as in hg.6 wilh linear g&i.&l be- io mM ammonium m m w pH 6.5 in w.1~1 lod 10 mM ammnium .ccmte pH 6.5 in socmniuile. W e n t : 0 1 0 Ik for 30 min.

I .c

0.8

0 E 2 0 . E t 0

0) 0 c 0 g 0.' n CI VI

0.2

Amino Acid Sequences of Chains of Earthworm Hemoglobin I

(4

6515

IO 20 30 Tirne(rnin1

Fig.9 Rechmmropaphy of peptide fraction, V4 + V6. isolated as shown in Fig.7. on 'IPLC. 7hc condition5 WCIC same 8s in Fig.8. Gradicnc 1025 40 for 30 -.

Table 11. Cham c: Amino acid c~rnp~si !mn of pcplidsr dcnvcd by clcavagc with crypin. V8 prolcarc and 0-idow, benmc acid. Valuer in pmnthcacr arc the number of residues determined by sequencing.

Fraction TI B (TZ) (T16) (TII-2) T3 T I TS T6 T7 T8 T9 TI0 +IO' TI1 T I I ~ I AI (Tl l -2) (T2) TI2 TI3 TI4

FraCtlO"

AEX 2.1(21 Thr 1.0(1) Scr I .O(I ) .23 0.23 GI= 4.3(41 1.10 1.10 a.9(1) I . I ( I )

1.8(2)

FTO

I . Z ( I ) 1.1(1) 3.7(4) 1.0(1) I.0(1)

2.1(2) 2.0(2) 1.1(1) 1.1(1) 1.0(1)

1.0(11 1.0(1) 0.07 2.1(2) 1.0(11 1.0(1) 1.0(1) 2.1(2) 1.1(1) 1.99

0.88 0.88 1.0(1) 1.0(1] 1.0(1) 0.96

0.14

0.9(1) 0.9(1) 0.03 1.16

0.9( I I

2 0.33 0.33 lncyr 1.4(2) 0.18 Val Met

0.18

~ ."

LC" I lC 0.84 0.84 1.0(1)

0.61 0.30 0.31 1.0(1) 0.97(11 1.0(1)

3.0(3) WI) 1.1(11 I.O(I) 1.0(11 0.9(1) 0.88

LO(]) 1.98

I 9 9

1.98

1.76

1.16

0.96

0.88

1 2 7

7.1(71 ASX Thr

0.9(11

Total 13 4 4 3 7 6 2 5 5 3 1 0 1 1 1 0 7 4 Position 1-13 14-17 138-14171-73 18-24 25-30 31-32 33-37 38-42 43-45 46-55 5666 57~M 67-73 67-70

3 4 32 5 4 Total

Yield(%) 14 21 22 17 27 30 5 5 4 12 23 18 11 Y>cld(B) 71-73 14-17 74-105 106-110 111-114Porit~on

TI5 TIS' TI6 TI7 TI8 TI9 V I V2 V3 V4 V5 V5' V6 V7 V 7 ~ 1 V7-2 V7-3 V8 V9 VI0 TWA4

2%" 115-331 116-137138-141 142-146 147-1S0 151-153 1-4 5-10 11-20 21-27 28-52 35-52 53-60 61-103 61-68 69-103 93-103 104-106 107-118 119-153 137-153POSillOn Yald(%) 9 I5 19 23 25 I 1 14 7 11 8 5 3 9 3 5 1 2 I s Y'e1d(4L1

23 22 4 5 43 3 4 6 10 7 25 18 8 43 8 35 I 1 3 12 35 l7 Total


Table 111 C h i n c: Sequcnct analyns of popndcs obwncd by clcavagc with ryyprin. V8 O ~ I C ~ S C and o-~doroknzoic acld

Table Ill. (conlmucd) Cham c Syvcncc MPYIII of pcpudcs obmncd by clcavagc wtth %sin. V8 pro~carc and o-~cdoroknmc acid

Allli"O Acid

I 2 3 4 5 6 7 8 9

I 1 12 I3 14 IS 16 17 I 8 19

21 22 23 24 25

1 2 3 4 5 6

I 2 3 4 5

I 2 3 4 5

I 2 3

I 2 3 4 5 6 7 8 9

10

I 2 3 4 5 6 7 8 9

11

I 2 3 4 S 6 7

I 2 3 4

I 2 3 4 5 6 7 8 9

I 1 12 13 14 I S 16 17 18 19

21 22 23 24 25 26 27 28 29 30 31 32

I 2 ?

10

20

10

10

20

4 S

D E H E H C C S E E D H R I V s Q D 1 L W R D

D T E s S

( K )

I G F G R

L L L T K

L A K

D I P D V N D L F

(K)

R V D I E H A E G P K

F S A H A L R F S A H

I L N G L D L A I N L L D D P P A L D A A L D H L A H s V R

E G V

s

16.3 14.6 6.6

4.6 2.7 3.0 2 2 9 4 8.2

4 3 3 6

8 1 14.1 7.4

11.4 7.2 2.3 3.2

11 8 8.1

2.1 1 3 4 4

6 3 3 6

2 7 3.2

14.0

130

4.0

6.4 3.9

5.4 4.6 8 1

10.5

7.7 0 5 8 3

2.3 1.1

10 3

0. I

4.0 4.8 9.1 2 3 1.9 0.9

n 1 0.7

n 02

17 0 13 9

13.8 7.8

13.6 15.8 12.6 1 1 8 6.8 4.7 4 8

2.8 0 9 0.7 1 6 1.1 1.8 1.7

6.4

6.0 3.8

7.5

20.7 47.3 24 5 12.3

23 6 36.2

32.5 32 5

29.4 19 3

25.2 27.2 11.2 13 7 10.0

12.1 11.3

9.9 11.8 19.4 12.9 10 5 7.9

15 5 8 3

7 3 5.0

12.8 5. I 9.0

10.9

1.0

13.9 6 7

6.5 2.9

D 1.8 T 2 5 E 4 0 E 1.8

D 2.S

D O S

D 0.4

E 2 0

E 2.8

~

I 2 3 4

1 2 3 4 5 6 7 8 9

I 1 12 13 14 I S 16 17 I8 19

21 22

I 2 3 4

I 2 3 4 5

I 2 3 4

1 2 3

I 2 3 4 5 6 7

I 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19

21

1 2 3 4 S 6 7 8

I 2 3 4 5 6 7 8 9

I 1 12 13 14 I S

I 2 3 4 5 6 7 8

I 2 3

In

2n

20

10

A H F K

F G E I L A T G L P

9 D D Y D A L A W K

S C L K

G I L T K

I s s R

L N A

I L W R D T E

S S K I K I G F G R L L L T K L A K D I P

L F K R V D I E

H A E G P K F s A H A L R I L

H A E G P K F S

V R E

24.1 14 6

9 4

2s.o 18.0 29 3 29.6

37 4 37 8

3 6

29 8 18.8

19.4

19.9 22.2 9.3

14 8 8.3

10.9

204

10 2

20.7 10.1 13.1 4.4 8.5

1.9 1.5 0.4 0.1

n.5 0 4

0.02

4 1

0.06

7.5 4.6 4.8

10.5

I9 6 I3 I 2 5

2.2 3.5 1 9 1.1 0 4 0 2 0 1

0 7 0 4 2.3 1 9

2 4 1 9

2 2 2 2

1 4 I .a 1.6 2 7 2 8

mace 0 3 0 s

0 2 0.3

0 6

face 0 4

2.9 3.8 2.6 2.2

0 3 I S

0 3 n l

1.03

I .2n

0.77 0.73 0.22

0.46 0.28 0.22 n 18

12.0

1.74 1.60

1.05

0.62 0 I8

0.28

7.8 4 5 4 9 2 2 0 3 0 2 01

8.3 12.3 10

Background ("mole$)

G 0.5

L 4.5 F 2.2 K 1.8 R 4.2 V 0.6 D 0.4 I 0.2

H 0.19 GO19

A 0.58

G 0 42 EO.50

P 0.41 K 0.28

F 0 . E A 0.36

A0.40

LO 18 I U.16

F 0.36

H 0.14

L 0.23

R 2.0

Amino Acid Sequences of Chains of Earthworm Hemoglobin

lnmoler) (nmolsr) Yield Background

v 9 I G (45) 2 V requcncn 3

4 8 5 6 H 7 F 8 K 9 K

10 F 11 G 12 E

TRPA4 I K (21) 2 S rcqucnccr 3 (C)

4 L 5 K 6 G 7 I 8 L 9 T

;: 7 13 S 14 R I5 L

27.9 29.8 32.8 34.7 34.8 22.8 11.4 18.7

19.9

28.3 10.2

28.8

7.6 1.7

6.4 3.3 3.2

8.3

4.0 4.1

1 3

4.2 1.3

5 5

2.7

8.2

0 7

3 0

Ammo Acid B y ryuencc B y BnalySil B y ana1ysi. ofchains o f V 8 pcptldcs

pro 5

3 Val 1/2cys

6 MCt 0 I lC 11 LC" 22

1 Phc 5

9 TIC 3

% 14

T F

2; 13

A;g 7

Total 153

19 7

3.9 6.4

19.7

3.9

15.1 162 6.5

5.9 8. I

14.3

5.3

14.2 2.2

6.3 6.0

8.0

n n 10 3 10 8 22 5 22.3

5.3 1.0 0.9

5.1 12.5 8 6

12 5 8.4

6.8 6.9

6517

of chemistry vol. no issue may 6502-6517,1988 1988 the u ... · the journal 0 1988 by the american...

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