Insect Biochem. Molec. Biol. Vol. 23, No. 3, pp. 421429, 1993 0965-1748/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

Characterization of Hemoglobin from the Backswimmer Buenoa margaritacea (Hemiptera:Notonectidae) Reveals High Electrophoretic Heterogeneity CHARLES R. VOSSBRINCK,*'t PAWEL A. OSMULSKI,:~ PETER G. DEBRUNNER§

Received 7 May 1992, revised and accepted 3 August 1992

Backswimmers (Notonectidae) are one of the three families of insects which contain species known to make hemoglobin. In backswimmers the hemoglobin allows the insect to maintain a prolonged neutral buoyancy during its dive. Sephadex G-200 column chromatography, SDS-polyacrylamide gel electrophoresis and HPLC demonstrate a molecular weight for the monomeric hemoglobin of Buenoa margaritacea of 15,000-19,000Da. u.v.-visible spectra of eight hemoglobin derivatives (FeZ+:deoxy, O2, CO, NO; Fe3+:HzO, O H - , CN- , imidazole) are given. Native gel electrophoresis of the cyanomet form shows nine bands, while isoelectric focusing shows 12. Bands isolated from preparative IEF gels form multiple bands when rerun, indicative of a change in aggregation status after isolation. Interactions among various hemoglobin components are seen using 2-dimensional "titration" electrophoresis.

Insect Hemoglobin Bug hemoglobin Buenoa margaritacea Anisops Backswimmer Notonectidae Respiration

INTRODUCTION

Because of its unusual function, the hemoglobin from the Notonectidae (Buenoa and Anisops) is of particular interest to us. In the Notonectidae, hemoglobin is used neither to grab oxygen in a low oxygen environment as with Gasterophilus (Keilin and Wang, 1946) and Chi- ronomus, nor to circulate oxygen to tissues as with Chironomus (Wigglesworth, 1965). Instead, the Notonec- tidae use hemoglobin to maintain neutral buoyancy (Bare, 1928; Miller, 1966). The presence of hemoglobin in the Notonectidae was first reported by Hungerford in 1922 for Buenoa margaritacea. He observed that the hemoglobin was contained in cells surrounding the tracheal trunks, located just below the ventral surface of the insect's abdomen. The bubble carried by the insect was located over the abdominal spiracles.

Bare (1928) used the term "hemoglobin cells" in describing these cells from Buenoa margaritacea, but Poisson (1926) referred to them as tracheal ceils

*Agricultural Entomology, University of Illinois, 147 PABL, 1201 W. Gregory Drive, Urbana, IL 61801, U.S.A.

tAuthor for correspondence. ~Department of Biochemistry, University of Illinois, 496 Roger Adams

Lab., 1100 W. California, Urbana, I1 61801, U.S.A. §Department of Physics, University of Illinois, 331 Loomis Lab., 110

W. Green, Urbana, I1 61801, U.S.A.

("cellules trach6ales"). Because of possible confusion between the terms "tracheal cell" and "tracheal end cell", a cell which produces tracheoles, we will refer to the hemoglobin containing cells of backswimmers as hemoglobin cells. Bare first equated the presence of hemoglobin in B. margaritacea with the ability of this organism to remain poised in the water. Bare made the logical conclusion that the function of the hemoglobin was to allow Buenoa to take a smaller bubble, thus achieving neutral buoyancy while allowing the insect to remain submerged for longer as if it had taken a much larger bubble. Therefore, unlike other bubble carrying aquatic insects, most comparably Notonecta, Buenoa does not struggle to remain underwater with a large bubble. This gives Buenoa, a predator, the advantage of easily maintaining its position in the water while hunting.

Miller (1966) added low percentages of carbon monoxide to the air above Anisops pellucens in tanks to preferentially bind the hemoglobin, thus eliminating the oxygen carrying function of the hemoglobin. This al- lowed Miller to examine parameters such as duration and depth of diving with the oxygen carrying capacity of hemoglobin blocked. As a result, the time Anisops remained neutrally buoyant was almost eliminated.

Synthesis of hemoglobin in B. confusa from amino acid precursors was first demonstrated by Bergtrom

421

422 CHARLES R. VOSSBRINCK et al.

et al. (1976) who showed that at least two of five hemoglobin electromorphs could be synthesized by the hemoglobin cells. Bergtrom (1977) also showed this hemoglobin to have a monomeric molecular weight of ca 14,000 Da.

Wells et al. (1981) showed a triphasic hemoglobin- oxygen equilibrium curve for A. assimilis, demonstrating the occurrence of cooperativity at high hemoglobin concentrations. They found four electromorphs using an isoelectric focusing column and demonstrated subunit aggregation (dimers, trimers, tetramers and pentamers were observed) of deoxygenated hemoglobin using cross- linking agents.

The purpose of this study is to examine some of the general biochemical properties of B. margaritacea hemo- globin. This information will serve as a basis for further study.

MATERIALS AND METHODS

Collection, identification and rearing

For purposes of biochemical analysis adult B. mar- garitacea were collected from the edge of a local pond (Champaign, Ill.). In addition, B. margaritacea were cultured in the laboratory in 10 gai aquaria. They were fed on Daphnia magna and given alfalfa (Medicago sativa) or smartweed (Persicaria hydropiper) stems in which to oviposit. Voucher specimens were put in 70% ethanol.

Initial purification

All procedures were done on ice or at 4°C. Approxi- mately 50-150 bugs were used per preparation. Using a pair of forceps the abdomen was separated from the thorax, and the gut and eggs were removed from the abdominal cavity. 20-25 of these abdomens containing the hemoglobin cells were homogenized in a 1.5 ml conical centrifuge tube in 200 #1 20mM Tris, pH 8.2 with an homogenizing pestle. The tubes were spun in a Beckman Microfuge E microcentrifuge (15,000 rpm) for 5 min and the supernatant containing hemoglobin was removed. The pellet was again homogenized after the addition of another 200/d of buffer to extract any remaining hemoglobin and centrifuged. The super- natants were pooled, centrifuged again to remove any remaining fat and cuticle, and applied to a Sephadex G-75 column (1.5 × 25 cm) in 20 mM Tris, pH 8.2.

Determination of molecular weight

Molecular weight of B. margaritacea oxyhemoglobin was determined in three ways: (1) By gel filtration chromatography on a 1.5 × 25cm Sephadex G-200 column (20mM Tris, pH 8.2), at a flow rate of 2 ml/h. Fractions were collected every 20 min and were measured at 230, 280 and 414 nm in an HP8452A diode array spectrophotometer. (2) By HPLC, using a Beck- man 110B solvent delivery system with a Beckman TSK 3000PW HPLC column (0.75 × 60cm) with 100mM potassium phosphate buffer, pH 7.0 containing 10%

ethylene glycol at a flow rate of 0. l0 ml/min. A Beckman 163 variable wavelength detector was used at 276 nm for protein detection. (3) The minimum molecular weight was determined by SDS-PAGE on a Mini-Protean Slab Cell Apparatus (Biorad). The stacking gel was 4% acrylamide (36.5:1 acrylamide:bis-acrylamide), 0.1% SDS, 125 mM Tris-HC1, pH 6.8. The resolving gel was 12% acrylamide (36.5:1 acrylamide:bis-acrylamide), 0.1% SDS, 375mM Tris HCI, pH 8.8. The running buffer was 25mM Tris, 192mM glycine, pH 8.3. Gels were run at 200 V for 45 min, at room temperature.

Ultraviolet-v&ible spectroscopy

The concentration of heme and thus of hemoglobin was determined for the purpose of calculating the millimolar extinction coefficient for the oxyhemoglobin derivative. This was accomplished by spectrophoto- metric measurement of freshly purified oxyhemoglobin pH 8.2, followed by acidification with a known amount of nitric acid to pH 3.2 and direct measurement of iron content with a Jarrell-Ash Plasma AtomComp model 975 ICP spectrometer. Derivatives were then made from known amounts of hemoglobin, based on the oxy spectra, and the absorbance maxima measured. The deoxy derivative was prepared by the addition of a 2-fold molar excess of sodium dithionite. The cyanomet form was prepared by the addition of a 2-fold molar excess of K3Fe(CN)6 followed by a 2-fold molar excess of KCN. The salts were then removed by 2 3 con- centrations through a Centricon 30 centrifugal con- centrating membrane (Amicon) or by Sephadex G-25 column chromatography. The CO derivative was pro- duced by passing 60-90 bubbles of carbon monox- ide through the cuvette containing the hemoglobin sample. The NO ferrous derivative was obtained by the addition of ascorbate followed by potassium nitrite under an atmosphere of nitrogen, as in Trittelwitz et al. (1972).

Native acrylamide gel electrophoresis

Native acrylamide gel electrophoresis was done using a modification of the procedure described by Riggs (1981). The B. margaritacea hemoglobin run on native gels was in the cyanomet form. The stacking gel con- sisted of 4% acrylamide, 0.11% bis-acrylamide with 125 mM Tris-H3PO4 gel buffer, pH 8.9. The resolving gel was 7.5% acrylamide, 0.2% bis-acrylamide with 375 mM Tris-HC1 buffer, pH 8.8. The cathodic electrode buffer was 52 mM Tris-glycine, pH 8.9, and the anodic buffer was 100mM Tris-HC1, pH 8.1. The gel was electrophoresed to the anode at 100 V for 2 h at room temperature. Subsequent to electrophoresis the gels were either stained for protein in 0.1% Coomassie brilliant blue R-250 (40% methanol, 10% acetic acid in water, v/v) for l h followed by destaining for l day in 40% methanol, 10% acetic acid in water, or bands were eluted from the gel and concentrated through a Centricon 30 centrifugal concentrator (Amicon) prior to being run on a second native gel.

CHARACTERIZATION OF HEMOGLOBIN FROM THE BACKSWIMMER 423

Isoelectric focusing

Samples were run on horizontal 10 cm gels (Pharma- cia flatbed FBE-3000) with broad range ampholytes (2% LKB Ampholine, pH 3.5-10). Best results were obtained when the samples were loaded from the acidic side. Gels contained 2 M urea, 5% (v/v) glycerol, 5% acrylamide, and 0.3% bis-acrylamide. Gels were prerun for 30 min at 12 W, loaded with B. margaritacea oxyhemoglobin and run at 12W for 1 h followed by 2.5h at 1000V. Gels were run at 4°C. Electrode strips soaked with 1 M H3PO 4 and 1 M N a O H were used in all the experiments. A preparative gel was run by loading a single sample along the entire width of the gel. The resulting bands were cut from the preparative gel, homogenized in a small amount of 20 mM Tris-HC1 buffer, pH 8.2 in a micro- centrifuge tube, spun at 15,000 rpm for 5 min and then concentrated through a Centricon 30 centrifugal concen- trator (Amicon). These purified bands were then rerun on IEF. Gels to be stained for protein were fixed in 10% TCA and stained in 0.04% Coomassie brilliant blue R-250 in 10% TCA, 50% methanol (v/v).

Titration electrophoresb

To investigate further the heterogeneity of B. mar- garitacea hemoglobin and the interactions between hemoglobin electromorphs, titration curves of hemo- globin produced by electrophoresis in a pH gradient of focused ampholites were used, as described by Rosengren et al. (1977) and Krishnamoorthy et al. (1978). A horizontal IEF gel was prepared as above and run at 6 W for 1 h followed by 1000V for 2 h to set up the pH gradient. The electrode strips were then removed from the gel and the gel was turned 90 °. A filter paper strip containing the hemoglobin sample was placed down the center of the gel parallel to the electrodes and 300 V was applied perpendicu- lar to the pH gradient. The various hemoglobin

0.8

0.6

Rf

0.4

0.2

0.0 1 04

(a) t.O

1 - cyt. c

~ 2w,~ 4 ; : 'Y;°ZoY:Ler 1 1

. . . . .

~ 6 ~ ® 7

105 molecular weight

(b) 29

Buenoa Hb 1 - cyt. c ~ z 9 . 28 " ( ~ " / 2 - lysozyme

_ ..~.1_ ~ j ~ @ 4 3 - Mb 4 - {x-chymotrypsin

2 7 - ~ 5 5 - Hb dimer 26 - ~ 6 - BSA

RT

[min]25123- 24-.. "

22 1 04 1 05

molecular weight

FIGURE 2. Relative molecular weights of B. rnargaritacea hemo- globin based on (a) SDS-PAGE and (b) Beckman TSK 3000 PW HPLC column chromatography. Vertical error bars indicate the standard deviation of at least three independent determinations. Size markers: cytochrome-c (12,400Da), lysozyme (14,400Da), human hemoglobin monomer (16,000Da), myoglobin (18,000Da), ~-chy- motrypsin (25,100 Da), human hemoglobin dimer (32,000 Da), BSA

(68,000 Da) and glucose oxidase (186,000 Da).

3

O O t - -

~ 2

..D

1

Buanoa Hb

molecular weight f

0

r ,*, Hb l - - A2ao

x I - - - A414

/ / ~ ', / (~ LMW

!, 5 1 0 1 5 20 25 30

elution volume [ml] 35

FIGURE 1. Elution profile of crude B. margaritacea hemoglobin from Sephadex G-200. HMW indicates a high molecular weight fraction collected in the void volume. LMW indicates low molecular weight compounds in the inclusion volume. Insert shows the relative elution volume of the hemoglobin compared to the four size markers used: cytochrome-c (12,400Da), myoglobin (18,000Da), ~-chymotrypsin

(25,100 Da) and human hemoglobin tetramer (64,000 Da).

components should, in principle, migrate depending on their charge and aggregation status at each pH along the gel.

RESULTS

Molecular weight

Figure 1 shows the elution profile of the crude sample run over a Sephadex G-200 column. In addition to the mare peak (B. margaritacea hemoglobin monomer) we observe a high molecular weight peak (HMW) absorb- ing at 414 nm, running ahead in the void volume. The low molecular weight peak (LMW) represents com- pounds present in the inclusion volume. The size esti- mate using Sephadex G-200 column chromatography shows that the B. margaritacea hemoglobin elutes between the myoglobin (18,000 Da) and cytochrome c (12,400Da) standards (see insert, Fig. 1). Estimates based on S D S - P A G E indicate a minimum (monomeric) molecular weight of 15,000 Da [Fig. 2(a)] while estimates

424 C H A R L E S R. V O S S B R I N C K et al.

125

100

E .'9 75 ,.? '5 E 50 E

25

(a)

r - - - de°xyHbl I '

0 2 4 0 340 4 4 0

1 6

. . . . , 0 540 640 740

~, [nm]

1 2

(c)

120

0 240

T E o

80 o E

co 40

- - metHb(H20) ] -~-..7 metHb(OH) ~

340 440

12

t

~t

540 640 740

160

120 ,'T E

-.% ,v 80

E E

(b)

q

HbCO

i _ _ _ HbNO

40

0 240 340

0 440 540 6J, O 740

~, [nm]

-16

12 7 E o

± O E E

120 (d)

t - \ t \ i x

o 540 640 740

- - metHb(CN) ] 1 00 - - - metHb(Irn) / , t ~

8O

60

40 - . ,

20

0 240 3 ;0 4 i 0

~, [nm]

[nm]

F I G U R E 3. Ul t raviole t -vis ible absorp t ion spectra of B. margaritacea hemoglob in derivatives. (a) and (b) represent l igands bound to hemog lob in in the reduced (Fe 2+) state, while (c) and (d) show l igands bound in the met (Fe 3+) state. (a) oxy and

deoxy, (b) CO and NO, (c) H20 and OH and (d) C N and imidazole.

14

• 12

• 10

8

6

4

2

based on HPLC indicate a molecular weight near 19,000Da [Fig. 2(b)]. The agreement in size estimates between SDS-PAGE and Sephadex G-200 indicates that the B. margaritacea hemoglobin is a monomer under our isolation conditions.

Ultraviolet-visible spectroscopy

Figure 3(a) (d) shows the spectra of eight bug Hb derivatives. The millimolar extinction coefficients and absorbance maxima are presented in Table 1. With the

T A B L E 1. Abso rbance m a x i m a and mi l l imola r ext inct ion coefficients o f the spectra for the

B. margaritacea hemog lob in der ivat ives shown in Fig. 3

2 ' 2 ,a, ,a, ), ), 2 Der ivat ive E * E ~ ~ ~ e E

Fe 2 +

Deoxy 270 345s 430 556 46.5 39.3 114 12.2

02 276 346 415 542 - - 578 - - 48.1 34.3 112 14.1 14.4

CO 274 346 421 539 571 39.4 27.9 144 13.8 12.4

N O nd 350 416 542 - - 576 - - nd 44.9 109 12.3 - - 11.2 - -

Fe 3+

H20 274 358s 410 530 572s 606s 634 37.8 36.1 119 10.6 6.3 2.23 2.12

O H 274 352s 412 538 574 606s - - 50.0 45.2 114 11.7 9.78 6.65 - -

C N 276 358 420 538 35.7 31.0 97.7 10.7

Im 264 362 414 536 568s - - - - 39.4 29.1 92.8 12.5 9.88 - - - -

" s " denotes the presence of a shoulder in the spectrum, the pos i t ion de te rmined on the basis o f the m i n i m u m in the second derivative. *2 = absorbance m a x i m u m in nm. tE = mi l l imola r ex t inc t ion coefficient.

CHARACTERIZATION OF HEMOGLOBIN FROM THE BACKSWIMMER 425

F IGURE 4. Separation of B. margaritacea cyanomethemoglobin by native polyacrylamide gel electrophoresis. Lane 1 shows the Sephadex G-75 purified sample of cyanomethemoglobin; lanes 2 7 represent the top six bands eluted and concentrated

from a separate native gel and rerun. The gel was stained with Coomassie brilliant blue R-250.

itil,iii~i !~ii ~iii ¸

FIGURE 5. Separation of B. margaritacea hemoglobin on an IEF gel: lane 1: oxyHb, lane 2: metHb, lane 3: cyanometHb. Stained with Coomassie brilliant blue. Unlabeled bands, those above band l, represent denatured protein missing the heme

and were not visible before Coomassie brilliant blue staining.

426 CHARLES R. VOSSBRINCK et al.

FIGURE 6. Unstained preparative IEF gel of B. margaritacea oxyhemoglobin.

F IGURE 7. IEF gel of bands previously separated on the preparative IEF gel shown in Fig. 6. Lanes 1-12 represent bands 1 12, respectively, cut from the gel in Fig. 6.

CHARACTERIZATION OF HEMOGLOBIN FROM THE BACKSWIMMER 427

except ion o f one spec t rum presented by Berg t rom et al. (1976), there have been no previous spectral da t a pre- sented for no tonec t id hemoglobin .

Native gel electrophoresis

Before s taining, a to ta l o f nine red hemoglob in bands are visible on the nat ive gel. S ta in ing with Coomass ie br i l l iant blue is necessary to visualize more clearly the b o t t o m two bands (Fig. 4, lane 1). However , af ter s taining, reso lu t ion is lost and the third and four th as

well as the fifth and sixth bands can no longer be d is t inguished as separate . The top six bands were cut f rom an uns ta ined nat ive gel, run on a second nat ive gel and s ta ined with Coomass i e br i l l iant blue (Fig. 4, lanes 2-7). Each band cut f rom the nat ive gel yields 1-2 adjacent bands .

lsoelectric focusing

Figure 5 shows the oxy, met, and cyanomet der ivat ives o f B. margaritacea hemoglob in run on an I E F gel. The

FIGURE 8. Results of "titration" electrophoresis of B. margaritacea oxyhemoglobin (a) gel unstained; arrow 1 shows the convergence of several bands at high pH and arrow 2 shows a band splitting near the line of sample origin. (b) Gel stained; arrow 1 shows a deviation of the titration curves indicating an interaction between bands, arrow 2 shows the line of sample

origin.

428 CHARLES R. VOSSBRINCK et al.

gel was stained with Coomassie brilliant blue. The three forms show similar banding patterns and spectrophoto- metric assays indicate that they are in their expected oxidation state with the ligand intact. Bands not labeled, above band 1, represent denatured hemoglobin in which the heme has popped out; these bands were not visible before Coomassie brilliant blue staining.

Figure 6 shows an unstained preparative IEF gel of B. margaritacea oxyhemoglobin. Twelve bands are visible on this gel. These bands were cut from the gel and rerun on a second IEF gel (Fig. 7). On this second gel, a large number of bands are seen in most of the lanes. As a control human Hb and sperm whale myoglobin were run under the same conditions. In this case, each band cut from the IEF gel gives rise to a single band when rerun on a second IEF gel (data not shown).

Titration electrophoresis

Figure 8(a) and (b) shows the total hemoglobin mix- ture run on the titration electrophoresis system described above. Figure 8(a) (unstained) clearly shows an intersec- tion of several bands at high pH (arrow 1) as well as an apparent band splitting (arrow 2) starting near the strip placement. Figure 8(b) (stained) shows clearly the effect two bands have on each other's mobility (arrow 1) at pH 7.5-8. Arrow 2 indicates the location of the filter paper strip containing the hemoglobin sample.

DISCUSSION

Bergtrom (1977) and Wells et al. (1981), observed a heme containing peak eluting in a tetrameric size range from the Sephadex G-200 column. Our data show a single heme containing peak, of monomeric size. This dissimilarity could be attributed to the use of a Tris buffer system with oxyhemoglobin rather than a phos- phate cyanide buffer or to differences in the hemoglobin among the species studied.

The molecular weight estimate of 15,000-19,000 Da for the hemoglobin monomer of B. margaritacea is similar to hemoglobin size estimates obtained for other species of backswimmers including B. confusa (14,000Da; Bergtrom, 1977) and Anisops assimilis (17,000 Da; Wells et al., 1981).

The use of the term "hemoglobin" vs "myoglobin" may be only a matter of convention where the inver- tebrate oxygen transport and oxygen storage pro- teins are concerned. As stated above, the hemoglobin of B. margaritacea is a monomeric protein, like myo- globin, under our isolation conditions. On the other hand, the B. margaritacea oxyhemoglobin visible spec- trum [Fig. 3(a)] shows a maximum at 415 in the Soret (compared to 415 for human hemoglobin vs 418 for sperm whale myoglobin) and a maximum of 578 for the

peak (577 for human hemoglobin vs 581 for sperm whale myoglobin; Antonini and Brunori, 1971).

The spectrum of B. margaritacea oxyhemoglobin exhibits a smaller (1.02) ~:/~ ratio (see Table 1) than that

seen for vertebrate hemoglobins (1.08; Antonini and Brunori, 1971). The most distinct difference is a large red shift from the value of vertebrate hemoglobin at 500nm (Antonini and Brunori, 1971) to 530nm for B. rnargaritacea hemoglobin in the aquamet form. An additional peak is seen at 634 nm which disappears when the pH is increased [Fig. 3(c)].

Human hemoglobin is phylogenetically more closely related to human myoglobin than to insect hemoglobin (Goodman et al., 1987). Our u.v.-visible data and the high degree of cooperativity shown by bug hemoglobin in oxygen binding studies (Wells et al., 1981), support the general designation of this protein as hemoglobin. This similarity of the B. margaritacea heme protein to vertebrate hemoglobin represents an instance of either convergent or parallel evolution.

Native gel electrophoresis of B. margaritacea hemo- globin reveals a substantially different pattern than that seen with IEF gel electrophoresis (Fig. 4, lane I and Fig. 6, respectively). With native gel electrophoresis we see nine bands, compared to 12 bands with IEF. Figure 4 shows further that when bands are sliced from the native gel, eluted and rerun, either a single band or two adjacent bands result (Fig. 4, lanes 2 7). Trewitt et al. (1981) obtained similar results for Chironomus thummi Hb in the same form (cyanomet) run on native gels. They found that when the original Hb band was eluted from the gel with phosphate-cyanide buffer and rerun on a second gel, additional, more electronegative bands were seen. When the 12 bands from IEF are sliced from the gel and rerun, however (Fig. 7), a large number of distinct bands arise. These bands are spectrophoto- metrically identical and thus do not represent different heme oxidation states. In contrast, when human hemo- globin and sperm whale myoglobin are run under the same conditions, each band cut from the first gel gives rise to a single band when run on a second gel. From this we infer that the component or components of the B. margaritacea hemoglobin bands are reaggregating after isolation from the preparative IEF gel and forming new bands on the second IEF gel.

Perhaps the most convincing evidence of protein interactions among the various B. margaritacea hemo- globin components is the result of the "ti tration" electrophoresis. Figure 8(a) and (b) shows total B. margaritacea hemoglobin run on this system, unstained and Coomassie brilliant blue stained, respectively. Figure 8(a) clearly shows a convergence of several bands at high pH as well as an apparent band splitting near the line of sample origin. Figure 8(b) shows more clearly an interaction between bands resulting in a deviation of the titration curves similar to that described by Righetti et al. (1978) for cytochrome b5 and methemoglobin. These observations strongly support the idea that this hemoglobin represents a complex mixture of interacting proteins.

Studies on C. thumrni hemoglobin show hetero- geneity due to differences in amino acid composition

CHARACTERIZATION OF HEMOGLOBIN FROM THE BACKSWIMMER 429

( B r a u n i t z e r a n d Braun , 1965), i nd i ca t i ng the p resence o f

m o r e t h a n one gene. D N A sequenc ing o f C. t h u m m i

h e m o g l o b i n revea ls a su rp r i s ing lack o f i n t rons in these

genes ( A n t o i n e and Niess ing , 1984). B iophys ica l s tudies

o f B. m a r g a r i t a c e a ( O s m u l s k i et al., 1992) sugges t t ha t

B u e n o a h e m o g l o b i n c o m p o n e n t s a re c o d e d fo r by m o r e

t h a n one gene. F u t u r e s tudies o f b u g H b m u s t i nvo lve

pur i f ied s ingle c o m p o n e n t s . In l ight o f the u n c e r t a i n t y

o f the a g g r e g a t i o n s ta tes o f h e m o g l o b i n on na t ive and

I E F gels in this s tudy, the best a p p r o a c h for the iso-

l a t ion o f p u r e h e m o g l o b i n c o m p o n e n t s m a y be the

exp re s s ion o f i so la t ed c D N A s in an a p p r o p r i a t e

exp re s s ion vec tor .

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Antoine M. and Niessing J. (1984) Intron-less globin genes in the insect Chironomus thummi. Nature 310, 795-798.

Antonini E. and Brunori M. (1971) In Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam.

Bare C. O. (1928) Haemoglobin cells and other studies of the genus Buenoa (Hemiptera, Notonectidae). Univ. Kans. Sci. Bull. 18, 265-349.

Bergtrom G. (1977) Partial characterization of haemoglobin of the bug, Buenoa confusa. Insect Biochem. 7, 313 316.

Bergtrom G., Gittelman S., Laufer H. and Ovitt C. (1976) Hemo- globin synthesis in Buenoa confusa (Hemiptera). Insect Biochem. 6, 595-600.

Braunitzer G. and Braun V. (1965) Zur Phylogenie des H/imoglobinmolekiils: Untersuchungen an Insekten-H~imoglobinen (Chironomus thummi). Hoppe-Seyl. Zeitschr. Physiol. Chem. 340, 88 91.

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Hungerford H. B. (1922) Oxyhaemoglobin present in backswimmer Buenoa margaritacea Bueno (Hemiptera). Can. Ent. 54, 262-263.

Keilin D. and Wang Y. L. (1946) Haemoglobin of Gastrophilus larvae. Purification and properties. Biochem. J. 40, 855-866.

Krishnamoorthy R., Bosisio A. B., Labie B. and Righetti P. G. (1978) Titration curves of liganded hemoglobins by combined isoelectric focusing-electrophoresis. FEBS Lett. 94, 319 323.

Miller P. L. (1966) The function of haemoglobin in relation to the maintenance of neutral buoyancy in Anisops pellucens (Notonectidae, Hemiptera). J. exp. Biol. 44, 529-543.

Osmulski P. A., Vossbrinck C. R., Sampath V., Caughey W. S. and Debrunner P. G. (1992) Spectroscopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera:Notonectidae). Biochem. biophys. Res. Commun. 187, 570-576.

Poisson R. (1926) L' Anisops producta Fieb. (Hemiptere: Notonectidae), Observations sur son Anatomie et sa Biologie. Archs Zool. exptl Gen. 65, 181-208.

Riggs A. (1981) Preparation of blood hemoglobins of vertebrates. In Methods in Enzymology (Edited by Antonini E., Rossi-Bernardi L. and Chiancone E.), Vol. 76, pp. 1 28. Academic Press, New York.

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Rosengren A., Bjellqvist B. and Gasparic V. (1977) In Electrofocusing and lsotachophoresis (Edited by Radola B. J. and Graesslin D.), pp. 165 171. de Gruyter, Berlin.

Trewitt P. M., Bergtrom G. and Laufer H. (1981) Cyanide-induced heterogeneity of Chironomus thummi Haemoglobins. Insect Biochem. 11, 167 172.

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