Polymorphic hemoglobin from a midge larva (Tokunagayusurika akamusi) can be divided into two different types

Biochimica et BiophysicaActa, 1157 (1993) 185-191 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

185

BBAGEN 23801

Polymorphic hemoglobin from a midge larva ( Tokunagayusurika akamusi) can be divided into two different types Mitsunori Fukuda, Takashi Takagi and Keiji Shikama BiologicalInstitute, Facultyof Science, Tohoku University,Sendai (Japan) (Received 15 October 1992)

Key words: Hemoglobin; Polymorphism;Distal. His; Midge (Tokunagayusurika) The hemoglobin from the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in eutrophic lakes in Japan, was composed of as many as 11 separable components (IA, IB, II, III, IV, V, VIA, VIB, VII, VIII, IX) on a DEAE-cellulose column. However, we have found that these components can be divided into two groups on the basis of their spectroscopic properties, one being named as the normal type (N-type) and the other being referred to as the low type (L-type). Since the major difference between them seemed to be the presence or absence of the distal (E7) histidine residue, which plays an important role in the stability properties of the bound dioxygen, the complete amino acid sequence was then determined for each typical component, namely, VII (N-type) and V (L-type): the former hemoglobin contained the usual distal histidine residue at position 64, whereas the latter one replaced it by isoleucine at position 66. The homology test for 40 N-terminal amino acid residues of all components also demonstrates that T. akamusi hemoglobin is composed of two different clusters showing a very early separation in the phylogenetic tree.

Introduction As for the midge (Diptera) hemoglobin, much attention has been directed to the proteins from some species of Chironominae, such as Chironomus thummi thummi [1-3] and C. thummi piger [4,5]: their hemoglobins are of low molecular mass, found in both monomeric and dimeric forms in the larval hemolymph, and show a high degree of polymorphism. However, Tokunagayusurika akamusi, a common species found in eutrophic lakes in Japan, belongs to a different subfamily (Orthocladiinae) from Chironominae, and its larva is quite unique in its morphology and ecological behavior [6]. It was therefore of interest to examine what kind of hemoglobin c o m p o n e n t s ' t h i s unique species has.

Correspondence to: K. Shikama, Biological Institute, Faculty of Science, Tohoku University, Sendai 980, Japan. Data supplementary to this article are deposited with, and can be obtained from, Elsevier Biomedical Press, B.V., BBA Data Deposition, P.O. Box 1345, 1000 BH Amsterdam, The Netherlands. Reference should be made to BBA/DD/531/23801/l157 (1993) 188-191. Abbreviations: Hb, hemoglobin; HbO2, oxyhemoglobin; Mb, myoglobin; metHb, methemoglobin; SDS, sodium dodecyl sulfate; CNBr, cyanogen bromide; FPLC, fast protein liquid chromatography; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); DACM, N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide; PMSF, phenylmethyisulphonyl fluoride.

In this p a p e r we first describe procedures for isolating hemoglobin components from the 4th instar larva of Tokunagayusurika akamusi by gel filtration on Sephadex G-50, followed by DEAE-cellulose chromatography. We then examine all the eluted components for their spectroscopic property in detail, and divide them into two groups. We have also determined the complete amino acid sequence o f the major protein of each group, and constructed a phylogenetic tree of T. akamusi hemoglobin components on the basis of their N-terminal 40 sequences. Materials and Methods

Chemicals Sephadex G-50 and G-75 were products of Pharmacia. DEAE-cellulose (DE-32) was purchased from Whatman. Reagents used for the protein sequencing were from Applied Biosystems Inc. All other chemicals were of reagent grade from Wako Pure Chemical (Osaka), and solutions were made with deionized and glass-distilled water. Isolation of hemoglobin components Frozen larvae (50 g) were thawed quickly and homogenized with a Teflon-glass homogenizer, in 1.5 vol. of 5 m M Tris-HCl buffer (pH 8.4) containing 0.! m M PMSF (Sigma) and 0.5 m M E D T A to prevent the

186 digestion caused by endogenous proteases. After insoluble material had been removed by centrifugation, the extract was fractionated with ammonium sulfate between 55 and 90% saturation. The crude hemoglobin solution thus obtained was then passed through a Sephadex G-50 column (5 x 90 cm) with 5 mM Tris-HC1 buffer (pH 8.4). The essential step was the chromatographic separation of the hemoglobin components on a DEAE-celullose column (4 x 16 cm) which had been equilibrated with 5 mM Tris-HCl buffer (pH 8.4). The elution was performed with a linear gradient of TrisHCI buffer from 5 to 150 mM at a constant value of pH 7.9. All these procedures were carried out at low temperature (4°C) as far as possible. The concentration of T. akamusi hemoglobin was determined, after conversion into cyanomethemoglobin, by using an extinction coefficient of 10.6 m M - 1 / c m -1 at 540 nm, the value being obtained on the basis of the pyridine hemochromogen method [7].

Spectrophotometric measurements Absorption spectra were recorded in a Hitachi twowavelength double-beam spectrophotometer (Model 557 or U-3210) equipped with a thermostatically controlled cell holder. Temperature was controlled by a water bath (Lauda thermostat K2 or Tamson TC3) maintained to within + 0.1°C.

Reduction, carboxymethylation and removal of heme Hemoglobin was precipitated by adding 10% (w/v) trichloroacetic acid, and the centrifugal sediment was dissolved in 6 M guanidine-HCl containing 0.2 M TrisHCI buffer (pH 8.5) and 10 mM EDTA. The protein was then reduced with 10 mM dithiothreitol and carboxymethylated with 15 mM iodoacetic acid. The reduced and carboxymethylated hemoglobin was dialyzed against distilled water, and heme was removed by extraction with acidic methylethylketone at -20°C.

Determination of the position of disulfide bridge The concentration of free cysteine residue was measured by the DTNB method, using an extinction coefficient of 13.6 m M - 1 / c m -1 at 412 nm [8]. To determine the position of disulfide bridges, noncarboxymethylated apoprotein (component V, 50 nmol) was first digested with pepsin in 20 mM HC1 at 37°C for 12 h. The peptide containing half-cystine residue, which was checked with a fluorescent dye DACM [9], was subjected to the sequence analysis to obtain PTHcystine.

Amino acid composition and sequence analysis The amino acid composition was analyzed with the ninhydrine method in a Hitachi 835-50 amino acid analyzer, or with the o-phthalaldehyde method in a Hitachi L-8500 analyzer. The amino acid sequence of peptides was determined by an automated sequencer 120A (Applied Biosystems model 477A). Results and Discussion

Characterization of T. akamusi hemoglobin components As shown in Fig. 1, the hemoglobin from T. akamusi was separated into as many as eleven components on a DEAE-cellulose column, these being named tentatively as IA, IB, II, III, IV, V, VIA, VIB, VII, VIII and IX according to the order of elution with a linear gradient of Tris-HC1 buffer from 5 to 150 mM at a constant pH value of 7.9. It should be noted here that the expanded scale is used for the concentration of the early eluted components of I to V, and that two major peaks V and VII were estimated approximately to occupy 15 and 30%, respectively, of the whole hemoglobin c o n c e n t r a -

i

i

V

' VII / I./, f // VIII

IA

'//- 150 ~6 I

1"

iV < )

-

iO0

'

Z-4

Enzymatic digestion and purification of peptides The apoprotein (20-50 nmol) was first cleaved with CNBr in 70% H C O O H at 25°C for 12 h. To obtain the overlapping of peptides, the whole protein (20-50 nmol) was also digested with staphylococcal V8 protease in 50 mM ammonium bicarbonate and with lysyl endopeptidase in 5 mM Tris-HCl (pH 9.0) containing 2 M urea, respectively, at 37°C for 4 h. In some case, peptic digestion was also employed. The peptides were purified by FPLC (Pharmacia) on a reverse-phase column, Cosmocil 5C18-AR (4.6 × 150 mm, Nakarai) or Aquapore RP-300 (4.6 x 220 mm, Applied Biosystems), with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 ml/min.

VIB1

-

11 ///

-

VIA

'IB

61

=x

I

50

~

~-

2

-0

(

300

500

Fraction Number

Fig. 1. DEAE-cellulosechromatographyof T. akamusi hemoglobin. Hemoglobin solution (250 ml, 500 mg) was applied to a DEAE-cellulose column (4 × 16 cm) equilibrated with 5 mM Tris-HCl buffer (pH 8.4). The elution was carried out with a linear gradient of Tris-HC1buffer from 5 to 150 mM at pH 7.9. The heme protein level was monitored by the absorbance at 417 nm (o), and the expanded scale was used until fraction number 325. Fraction size, 10 ml:

187 tion in polyacrylamide gel electrophoresis according to the specifications by Riggs [10]. Fig. 2 shows the typical absorption spectra of T. akamusi Hb, represented by two major components VII and V, in the oxy- and acidic met-forms. As for component VII (Fig. 2a), the a-peak of the oxy-form was always higher than the/3-peak with an absorbance ratio of a / f l being 1.06. In the Soret region, the y-peak of the acidic met-form appeared to be much higher than that of the oxy-form. These spectral features are almost identical to those of the usual mammalian myoglobins and hemoglobins [11], and are common to all the components of VI to IX. Therefore, this group was named as the normal type (N-type). As to component V (Fig. 2b), on the other hand, the a-peak was much lower than the fl-peak with the absorbance ratio being 0.82, so that this species was referred to as the low type (L-type). Furthermore, it is interesting that the Soret peak of its acidic met-form showed a profound blue shift accompanied by a marked decrease in intensity, as compared with that of component VII of the N-type. This spectral feature is very ,

1.5

VII ( N - t y p e )

(a)

/'~

o.15

I'

/ iI

1.0

O.lO

/ I

I 0.5

I

0.05

/

,fN

i 300

t 400

i

500

600

700

W a v e l e n g t h (nm)

1.5

i V (L-type)

i

(b)

o.15

1.0

0.10

0.5

).05

E .a <

i 300

i 400

i 500

600

700

Wavelength (nm)

Fig. 2. Absorption spectra of T. akamusi hemoglobin components. The typical spectra were represented by components VII (a) and V (b) both in the oxy-form(continuous line) and the acidic met-form (broken line). The concentration was 10 /~M for each in 0.1 M buffer; the pH was 7.9 for HbO2 and 7.2 for metHb, except that the complete acidic met-form for component V was obtained at pH 5.5. In contrast to the normal spectra for HbO2, the a-peak was always lower than the/3-peak for all the components I to V.

similar to that of Aplysia myoglobin lacking the usual distal histidine residue [12-14], and was common to all the L-type components (I to V). Other properties can also divide the hemoglobin from T. akamusi into two groups. For instance, the p K a value for the acid-alkaline transition of ferric met-form was one of the parameters that differentiated between the N- and L-type. From the spectrophotometric titration method monitored at 577 nm, the p K a value was obtained to be about 8 (ranging from 8.3 to 8.5) for N-type components, whereas it was about 7 (ranging from 6.9 to 7.4) for the L-type, except for component III bearing a higher value of p K a = 8.2. In SDS-polyacrylamide gel (12.5%) electrophoresis, all the components of T. akamusi Hb showed almost the same mobility indicating a molecular mass of about 15 kDa. However, the N-type components were found to exist in a dimeric form on the basis of the gel filtration pattern on a Sephadex G-75 column in 5 mM Tris-HCl buffer at pH 8.4. At any rate, one of the most remarkable features between the two types of T. akamusi hemoglobin seems to be the difference in intensity of the Soret peak of the acidic met-form (Table I). Recently, Shikama and Matsuoka have proposed that an absorbance ratio of the Soret peak of the acidic met-form to that of the oxy-form, namely the Ym~t/Yoxy-ratio, can provide a simple criterion for predicting whether or not a myoglobin (or a hemoglobin) has the usual distal histidine residue; the values higher than 1.0 are found for the usual type of myoglobins such as sperm whale Mb, whereas those of less than 1.0 are the ratio for the myoglobins lacking the usual distal histidine residue, such as Aplysia Mb [11]. In this respect, it is quite clear that the major component VII of T. akamusi Hb has the value of 1.19 belonging to the usual type. On the other hand, the Ym~t/Yo,~-ratio of component V was found not to be higher than 1.0, this suggesting strongly that the protein belongs to the Aplysia-type myoglobin. All of these results seemed to indicate that the major difference between the L- and N-type component is in the presence or absence of the usual distal histidine residue. To confirm this prediction, therefore, we have determined the primary structure of T. akamusi hemoglobin, laying special emphasis on those of the components V and VII.

Primary structures of T. akamusi hemoglobin components All procedures used to establish the complete amino acid sequences of component V and VII are summarized in Figs. 3 and 4, respectively. The proteins were cleaved with staphylococcal V8 protease, lysyl endopeptidase or pepsin, as well as with CNBr, and the resulting peptides were subjected to the automated

188 peak of the acidic met-form, component VII contained histidine at position 64, which may be assigned to the usual distal (E7) histidine residue, whereas component V replaced it by isoleucine at position 66, which may correspond to the Ell-lie revealed in the component III of Chironomus thummi thummi hemoglobin by Xray analysis [15]. Furthermore, the sequence homology between the component V and VII of T. akamusi hemoglobin was only 27% with different hydropathy profiles [16], suggesting that both proteins have differ-

Edman degradation. The detailed results including the elution profile of the peptides on a reverse-phase column and their amino acid sequences are all deposited in as the supplementary data. Component V was composed of 152 amino acid residues and has a calculated molecular mass of 17 197 Da for the apoprotein. Component VII, on the other hand, consisted of 150 amino acid residues with a calculated molecular mass of 16524 Da. As already predicted from the spectroscopic properties of the Soret

1 i0 20 Ala Phe Val Gly Leu Ser Asp Ser Glu Glu Lys Leu Val Arg Asp Ala Trp Ala Pro Ile (intact) ----9

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3O 4O l l i s GIy Asp Leu S l n G l y Thr A l a Ash Thr V a l Pile T y r Asn T y r Leu Lys Lys T y r Pro (intact) K1 ----9

---9

5O 6O Ser Ash Gln Asp Lys Phe Glu ~q*r Leu Lys Gly llis Pro Leu Asp GIu Val Lys Asp Thr ( intact ) ----9

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70 80 Ala Ash Pho Lys Leu Ile Ala Gly Arg Ile Phe Thr Ile Phe Asp Asn Cys Val Lys Ash K2 ----9

---9

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9O i00 Val Gly Ash Asp Lys Gly Phe Gln Lys Val Ile Ala Asp Met Set Gly Pro His Val Ala K3 K4 CNIV3 ---9

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130 140 Leu Asp Ser Thr His Gly Ala Ala Trp Asn Lys Met Met Asp Asn Phe Phe Tyr Val Phe K4 K5 ---9

----9

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---9

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CN3

----9

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CN4

150 K~r Glu Cys Leu Asp GI¥ Arg Cys Ser Gln Phe Ser -----)

~

~

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---9

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----9

CN4 ----9

Pl ----9

----9

Fig. 3. Summary of data used to establish the complete amino acid sequence of T. akamusi hemoglobin component V. Automated Edman degradation ( ~ ) was employed for the sequence determination of the peptides: K, lysyl endopeptidase peptide; V, staphylococcal V8 protease peptide; P, peptic peptide; CN, the peptide obtained by cyanogen bromide cleavage.

189

invariant Phe at CD1 and as the heme-binding proximal histidine at F8, respectively. Among all the components of T. akamusi Hb, only

ent heme environment. In these alignments, Phe-46 and His-98 of the component V, as well as Phe-44 and His-99 of the component VII, were placed as the 1 Asp Pro Thr (intact)

Trp

5 Val

Asp

Met

Glu

Ala

i0 Gly

Asp

Ile

Ala

Leu

15 Val

Lys

Ser

Set

Trp

20 Ala

K1 ---) ---) ---9 CNI ----9 ~

---) ---9 ~

---9

25 30 35 40 Gln Ile His Asp Lys Glu Val Asp Ile Leu Tyr Asn Phe Phe Lys Ser Tyr Pro Ala Ser (intact) ---9 ---9 --~ ---9 ---9 ----9 --~ ---9 ---9 ~ ---9 --9 --9 ----9 ---9 ~ ---9 ---9 ---9 K1 ---9 ---9 ----) ---) V1 ----9 ----) ---9 ---9 ---) ---9 ---9 ---9 ---9 ~ ---9 ~ ---9 ---9

Gln Ala Lys (intact)

Phe

45 Set

Ala

Phe

Ala

Gly

50 Lys

Asp

Leu

Glu

Ser

55 Leu

Lys

Asp

Thr

Ala

60 Pro

----9 ---9 ~

Vl

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65 Phe Ala Leu His Ala K4 ---9 ----9 ---9 ----9 ~

V2 ----9 ---9 ----9 ~

Val K4

Ala

Glu

Asn

Thr

Arg

Ile

Val

70 Set Val

Ile

Ash

Glu

75 Ala

Ile

Ala

Leu

Met

80 Gly

---9 ---9 ---9 ---9 ---9 ----) ---9 ----9 ---) ---9 ----9 ---9 ----9 ---9 ----9 CN2 ---9 V3 ----9 ---9 ---) ---9 ---9 ---9 ---9 ---9 ~ ---9 - - 9 ----) ---9 ~ ---9

85 Arg

Pro

Ala

Leu

Lys

90 Asn Val Leu K5 ----9 ----9 ~

Lys

Gln

95 Gln

Gly

Ile

Asn

His

i00 Lys

--~

CN2 ----9 ---9 ~

---9 ----9 ~

----9 ~

---9 ---9 ---o ---9 ---o ----9 ---9 ~

---9 ---9

105 Thr

llis P h e

ii0 Glu

Ala

V3 ----9

Gly K6

Arg

Gly

Val

Ala

Ala

---9 ---9 ----9 --~ ---9 ~ CN2 ---9 ---9 ---9 ----9 ---9 ---9 ~

Glu

Phe

115 Ala

Glu

Thr

---9 ---9 ----9 ---9 ~

--9

---9 ---9 ---9 ---9 ---9

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~

~ V4

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Leu

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Set

His

Ala

CN2 ---9 --9 ---9 ~ V4 V5 ---9 ---9 ---9 ~

Ash K7

CN2

Met

Tyr

Ser

125 Set

Gly

130 Gly

Tyr

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Ala

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~

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145 Val

Phe

150 Leu

Val

Pro

Glu

Thr

Lys

Lys

120 Leu

---) ---9 ---9 ----9

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GIu K6

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----9

135 140 Ala Trp Asp Ser Ala Phe Ash K7 ----9 ----9 ---9 ---9 ----9 ----9 ---9

----9 ---9 ---9 ---9 ---9 ----) ----9 ~

----9 ---9

CN3 ---9 ---9 ---9 ~

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V5

Fig. 4. S u m m a ~ of data used to establish the complete amino acid sequence of Z a k a m ~ i hemoglobin component VII. The procedures employed were the same as shown in Fig. 3 ~ r component V.

190 1

i0

50

20

40

44

t TA-IA TA IB TA-II TA-IV TA-V TA-III TA-VIA TA-VIB TA-VII TA-VII! TA-IX

A A D A A DP DP DP DP

TP T T V V

DADTQA D A D T QA TQ G EV D T E D E E K S D SEEK

K L L L

V K R R

I KA A A I HR I HG NK-

KAX S S W DAW DAW

~

D~E S G D I A L V K S S W D ESGDIALVKSSW ~D~E A G D 1 A L V K S S W ~N~D

A S

G G G G - -

V

DK-

A L V K S S W

-

-

DK

~

-

-

A ET T

Y LSlKIYIPIAIN ~1 E

A N ~" v

v~

KIKIYFI.,,I~ QIQ Y L KKI.EIYLY.JsNIK._qJD

A NZ v v~LYNE~FsY

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IE V D I L Y N F FIg S Y IE V

D

I

L

Y N

F

FIK

S

A

DK

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Y

A

NR

~

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:E V D I L Y N F FIK S Y

X

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E~'DILYNFF~XNY 30

20

10

z x LFIFIPIA ° , , , , , F QID ,

s s s z

A 40

42

Fig. 5. A possible alignment of T. akamusi hemoglobincomponents at N-terminal amino acid sequence. The residues identical in each cluster are boxed in, and the ones invariable in all components are marked with an asterisk (*). X, unidentified.

component V contained three cysteine residues; the one was found to be free by the thiol titration with DTNB, so that the remaining two would form an intra-molecular disulfide bridge because the component V exists in monomeric form. In fact, the sequence analysis of the P1 peptide followed by the cleavage with thermolysin clearly demonstrated that component V has a disulfide-bridge between Cys143 and Cys148 in a very close position as shown in Fig. 3. Such a disulfide bridge has not yet been reported in other midge larval hemoglobins. A matrix analysis was then carried out to test the sequence homology of the components V and VII with other midge hemoglobins sequenced so far, i.e. 10 components from C. t h u m m i t h u m m i [17-23], and 3 components from C. t h u m m i piger [5]. To these midge globins, the component VII showed higher degrees of similarity (40-48%), whereas the component V showed considerably low values (26-27%), suggesting that the L-type components might be a hemoglobin quite specific to the genus Tokunagayusurika and may have a different origin.

A phylogenetic tree o f T. a k a m u s i hemoglobin components The sequence of 42-44 N-terminal residues was easily determined for all the components of T. a k a m u s i hemoglobin by an automated sequencer, since these proteins were not blocked at N-terminus (Fig. 5). The similarity of each component was then calculated according to an unweighted pair-group clustering method [24], and is represented by a phylogenetic tree in Fig. 6. In this figure, all the N-type components (VIA, VIB, VII, VIII and IX) were found in the same cluster, and showed a very high degree of homology with each other (in more than 80% identity). The other cluster consisted of the L-type components (IA, IB, II, IV and V, except III), but they showed a wide variety of similarity (35-90%) with each other. Since the sequence homology between two clusters was found to be very low (20-30%), it may be concluded that T. akamusi has two origins of hemoglobin molecule: The N-type components may originate from the same ancestor as the dimeric components of Chironomus hemoglobin, while the L-type components may have

TABLE I Spectroscopic parameters of the two hemoglobin components from T. akamusi Absorption maximum(nm) (extinction coefficient(mM-1 cm x)) oxy-form T. akamusi V T. akamusi VII

acidic met-form 7". akamusi V T. akamusi VII

a 579.2 (11.0) 577.6 (14.1)

/3 546.8 (13.4) 542.0 (13.3)

y 413.2 (106.3) 416.0 (122.3)

UV 280.0 (41.2) 280.0 (42.4)

a//3 0.82

y/uv 2.58

1.06

2.88

CT~ 640.8 (2.7) 632.4 (3.8)

CT 510.4 (12.1) 501.2 (9.9)

y 396.8 (106.5) 406.4 (145.4)

UV 279.2 (36.3) 280.0 (40.4)

CT/CT ~ 4.48

y/uv 2.93

2.61

3.60

The measurements were carried out under the same conditions as in Fig. 2. CT: charge transfer band.

191

Acknowledgement

oo23VIA [~

VIB 0~ VII

N-type

0~[ t - ~ - IX

"~f

0121III

o.~ IB

0741II

+ ~0173

W e a r e i n d e b t e d to Dr. T. I w a k u m a , T h e N a t i o n a l I n s t i t u t e for E n v i r o n m e n t a l Studies, for his identifying s p e c i m e n s o f t h e m i d g e l a r v a e used. W e also t h a n k A. N a k a m u r a , S. O k a m o t o a n d Dr. T. F u r u k o h r i , D e p a r t m e n t o f Biology, K o c h i University, for t h e n i n h y d r i n e analysis.

L-type

References

IV V

Fig. 6. A phylogenetic tree of eleven components of T. akamusi hemoglobin. The tree was constructed from the Poisson-corrected values for the amino acid replacement, which are given for each branch, by an unweighted pair-group clustering method [24]. Standard errors at the branching points (a-j) were 0.011, 0.013, 0.032, 0.032, 0.027, 0.091, 0.029, 0.072, 0.076 and 0.033, respectively, and are represented by the hatched boxes. a n o t h e r origin. A t this point, it is i n t e r e s t i n g to n o t e t h a t c o m p o n e n t I I I ( L - t y p e ) s h o w e d a small d e g r e e of s e q u e n c e h o m o l o g y (less t h a n 25%) with o t h e r L - t y p e c o m p o n e n t s . R a t h e r , it shows a very high d e g r e e o f h o m o l o g y ( m o r e t h a n 7 5 % ) with t h e N - t y p e c o m p o nents. T h e r e f o r e , this m i n o r c o m p o n e n t I I I m a y occur f r o m a N - t y p e c o m p o n e n t by p o i n t m u t a t i o n f r o m histidine to a n o t h e r a m i n o a c i d r e s i d u e at t h e distal (E7) position, a n d this is o n e o f t h e r e a s o n s why c o m p o n e n t I I I s h o w e d a p K a v a l u e very close to t h o s e o f t h e N - t y p e c o m p o n e n t s in t h e a c i d - a l k a l i n e transition of t h e m e t - f o r m . In conclusion, t h e h e m o g l o b i n f r o m T. akamusi shows a very high d e g r e e o f p o l y m o r p h i s m , b u t its e l e v e n c o m p o n e n t s c a n b e d i v i d e d into two groups, a n o r m a l (N) a n d a low (L) type, by t h e i r a b s o r p t i o n s p e c t r a as well as t h e i r a m i n o acid sequences. T h e m a j o r d i f f e r e n c e was t h e lack o f t h e distal (E7) histid i n e r e s i d u e in t h e L - t y p e c o m p o n e n t s . T h e r e a r e s o m e l i m i t a t i o n s in j u d g i n g f r o m t h e N - t e r m i n a l sequences, w h i c h is n o t fully r e p r e s e n t a t i v e of t h e w h o l e p r o t e i n s , b u t t h e two t y p e s o f h e m o g l o b i n s e e m to have a d i f f e r e n t origin o r a very early s e p a r a t i o n in its phylogenetic tree, and the N-type components may result f r o m m u c h m o r e r e c e n t g e n e d u p l i c a t i o n s t h a n the L-type components.

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