Structure of the polypeptide chain of extracellular hemoglobin from the nematode Ascaris suum

Comp. Biochem. Physiol. Vol. 99B, No. 2, pp. 425-429, 1991 Printed in Great Britain

0305-0491/91 $3.00 + 0.00 © 1991 Pergamon Press plc

STRUCTURE OF THE POLYPEPTIDE CHAIN OF EXTRACELLULAR HEMOGLOBIN FROM THE NEMATODE ASCARIS SUUM SALEH DARAWSHEand EZRA DANIEL Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel (Tel: 03 5459749) (Received 4 December 1990)

Abstract--1. Ascaris suum extracellular hemoglobin is composed of eight identical single polypeptide chain subunits carrying two heme binding sites each. 2. Limited trypsinolysis followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a major band corresponding to half the molecular mass of an intact subunit. 3. Peptide mapping of tryptic hydrolysates yielded 27 to 30 fluorescamine positive spots, about half the number of lysyl and arglnyl residues in a polypeptide chain. 4. The findings indicate that a subunit of Ascaris hemoglobin consists of two structural units of roughly equal size, corresponding to two recurring sequences, connected together by the continuity of the polypeptide chain.

trifuged at 153,000g (ray 5.9cm) for 6hr and the supernatant containing excess hemin was discarded. The precipitate was dissolved in 50 mM NaCI/25 mM Tris-HCl buffer, pH 7,5, and dialyzed against 11 of the same Tris buffer. The dialyzed solution was centrifuged for 20 min at low speed and the supernatant was subjected to a second centrifugation at 153,000g for 6hr. The pellet containing heme-saturated hemoglobin was dissolved in 100gl Tris buffer and kept at 4°C until use. Concentrations of heme-saturated Ascaris oxyhemoglobin were measured by absorption spectroscopy. For absorption in the Soret (412 nm), a value for the extinction coefficient in 0.1 M phosphate buffer at pH6.8 of I I0,000M -l cm -~heme, determined from heme content and dry weight measurements, was used. Combined with a molecular mass per heine of 21.6 kDa (Darawshe et al., 1987), this corresponds to an absorption coefficient, at the wavelength of 412 nm, of A tcm t'/" 53.

INTRODUCTION A m o n g nematode extracelluar hemoglobins, that from Ascaris suum has been the most extensively studied (Lee and Smith, 1965; Wittenberg et al., 1965; Okazaki et al., 1965; Okazaki and Wittenberg, 1965; Darawshe et al., 1987). Structural investigations have shown that Ascaris hemoglobin, an 11.7 S molecule of 114",332,000, is composed of eight identical single polyepeptide chain subunits (Okazaki et al., 1965; Darawshe et al., 1987). In a previous study, evidence was presented that in hemoglobin isolated from Ascaris hemolymph, the binding capacity for heme is not fully realized (Darawshe et al., 1987). Titration with heroin showed the presence of two binding sites for berne per polypeptide chain of M r 41,600. In the present communication, two issues are addressed. The first is related to the ability of the extraneous heme, in berne-saturated hemoglobin, to bind oxygen reversibly. The second concerns the structure of the polypeptide chain, particularly whether the presence of two heme binding sites implies the presence of repetitive sequence in Ascaris hemoglobin.

Limited trypsinolysis

A 10 #1 portion of 0.45 mg/ml TPCK-trypsin in HCI, pH 2.5, was added to 200 #1 of 0.9 mg/ml solution of Ascaris hemoglobin in 0.05 M borate HCI buffer, pH 8.2, and the mixture was incubated at 37°C. A 30#1 aliquot was removed after 6 rain and at various time intervals thereafter, and added to an Eppendorf tube containing 15 #1 of 0.01 M phosphate buffer, 2% (w/v) in sodium dodecyl sulphate (SDS) and 2% (v/v) in 2-mercaptoethanol. The mixture was heated at 100°C for 2 min and a sample (25--40#1) was used for electrophoresis. This was performed on 5% polyacrylamide gels as described by Weber et al. (1972) in the presence of SDS. Protein bands were visualized by staining with Coomassie Blue.

MATERIALS AND METHODS

Ascaris hemoglobin was prepared as previously described (Darawshe et al., 1987). The apoprotein was obtained by removal of the heine moiety using the methyl ethyl ketone method (Teale, 1959; Ascoli et al., 1981). TPCK-trypsin (L-1-tosylamido-2-phenyl-ethyl chloromethyl ketone treated enzyme) was obtained from Sigma; TLC (thin layer chromatography) plates were from Merck.

Peptide mapping

Preparation o f heme-saturated oxyhemoglobin

To a solution containing about 1.2 mg hemoglobin in 3.5ml of 0.1 M phosphate buffer pH6.8 (A412/A2so-~ 1), heroin in two-fold excess of the amount needed for saturation w a s added. Ten rain later, a small amount of pure sodium dithionite was also added. The solution was cen-

Thin layer peptide mapping of Ascaris hemoglobin was carried out according to a protocol based on the procedures described by Huang and DeLange (1971), Gracy (1977) and Allen (1981). Hemoglobin (~600 #g) in a solution (200#1) containing 6 M guanidinium chloride/0.2 M Tris/0.1 M 2mercaptoethanol, pH 8.2, was incubated for 9 hr at 45°C. Iodoacetic acid in a 10-fold molar excess to 2-mercaptoethanol was added in the dark under nitrogen at 45°C and

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SALEHDARAWSI~and EZRADANIEL

the solution was maintained at pH 8.2 by the addition of 1 M NaOH. After standing for 15 min, 2-mercaptoethanol in 10-fold molar excess to iodoacetic acid was added, and the solution was dialyzed against 0.1 M NH4HCO 3 (five changes of buffer) at 4°C for 70 hr. The solution was lyophilized and the reduced carboxymethylated hemoglobin was redissolved in 130/~1 of 0.1M NH4HCO3/0.1mM CaCI2. Freshly prepared TPCK-trypsin (3/~g in 6 #1) was added and digestion was allowed to proceed for 12 hr at 37°C, at which time additional trypsin (3/Jg) was added. After 26 hr of total incubation, the solution was lyophilized. The lyophilized tryptic digest was dissolved in 2% (v/v) NH4OH (10 #l) and 7 #1 were spotted onto 'the origin of 20 x 20cm silica gel TLC plate. Electrophoresis in one direction was performed at pH4.4 in pyridine/acetic acid/acetone/water (2 :4: 15: 79) (v/v) for 4 hr at 440 V. The plate was dried and ascending chromatography was carried out along the second direction in pyridine/acetic acid/ 1-butanol/water (10:3:15: 12) (v/v) for 7 hr. The plate was then dried and sprayed with a solution of 0.1 mg/ml fluorescamine in acetone and viewed under long wavelength ultraviolet light. RESULTS

Absorption spectrum o f heme-saturated hemoglobin

The absorption spectrum of heme-saturated Ascaris oxyhemoglobin is presented in Fig. 1. Maxima of absorption occur at 280, ~345, 412 (Soret), 538 and 573 nm. The ratio of the absorbance at 412 n m to that at 280 n m is 2.1. A noticeable feature of the spectrum is that, in contrast to other hemoglobins (Di Iorio, 1981; Ilan and Daniel, 1989), the band at 573 n m has a lower intensity than the 538 n m band. Addition of dithionite to the oxyhemoglobin resulted in a deoxy derivative spectrum (also shown in Fig. 1) with maxima at 429 and 555 nm. U p o n removal of dithionite by dialysis the oxy spectrum was recovered, showing that the extraneous heine added to achieve full saturation is capable of reversible oxygen binding. Absorption o f apohemoglobin

Solutions of Ascaris apohemoglobin showed considerable light scattering. By assuming that the

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Fig. 2. Absorption spectrum of Ascaris suum apohemoglobin corrected as described in text for light scattering ( ) and of a mixture containing tryptophan, tyrosine and phenylalanine in the same ratio as in the protein (O). The upper and lower abscissae refer to protein and amino acid mixture, respectively. For easy comparison, the two spectra were normalized at their respective peaks. The aromatic amino acid content of a polypeptide chain of Ascaris hemoglobin, M, 41,600, has been taken to be 18 phenylalanines, 13 tryosines and 6 tryptophans (Wittenberg et al., 1965; Darawshe et al., 1987). Absorption coefficients for the three aromatic amino acids were those reported by Metzler et al. (1972).

residual absorption at wavelengths 2 > 350 n m was entirely due to light lost by scattering and taking into consideration a linear dependence of the intensity of scattered light on 2 -4 , the absorption spectrum of apohemoglobin was corrected and compared to that expected for a mixture of tryptophan, tyrosine and phenylalanine in the same ratio as in the protein (Fig. 2). A satisfactory fit of the apoprotein spectrum to that of the mixture was achieved by shifting the latter by 2 n m to the red. Good agreement was also found in the values of the absorption coefficients at the absorption maxima: Alcm 1% 12.0+0.5 for apohemoglobin at 278 n m compared with 12.1 for the amino acid mixture at 276 nm. The availability of Ascaris hemoglobin preparations having partially saturated heme binding sites (Darawshe et al., 1987) provided an indirect way of getting at the absorption coefficient of the apohemoglobin. Use was made of the equation: (A 1% , ~m)aoo + fi(A 1% lcm)obs = (A]*/' lem)h=m=

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Fig. 1. Absorption spectrum of Ascaris suum heinesaturated hemoglobin. ( ) oxy derivative; (. . . . . ) deoxy derivative. Deoxygenation was carried out by sodium dithionite. Solvent: 0.I M Tris-HCl, pH 7.5.

(1)

where fi is the average n u m b e r of hemes bound per polypeptide chain, (A)era)oh, )~ is the observed absorption coefficient for hemoglobin (0 < fi ~< 2), ( A l)% cm)apo is the absorption coefficient for apoprotein (fi--0), and (A 1% " the increment absorption coefficient ~¢m)hemeIS per bound heme. A plot of (A l'~/m)o~at 280 n m versus fi is given in Fig. 3. A linear extrapolation to fi equal to zero gave a value of 12.5 for ( A '°/' i cm )apo' Limited trypsinolysis Ascaris hemoglobin was exposed to the action of trypsin at pH 8.2, and products of trypsinolysis were analysed by SDS--polyacrylamide gel electrophoresis. The electrophoretic pattern of hemoglobin that had not been exposed to trypsin shows a strong band with

427

Structure of `4scaris hemoglobin I

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partially saturated with heine, fi and A I'~ were determined from heine content and dry weight measurements. Straight line through the experimental points (0) has a slope of 6.5 and an intercept of 12.5. For comparison, the value of `41'/. actually determined for apoprotvin (O) is lcm indicated.

mobility very close to that of ovalbumin (M, 45,000) and two faint bands of lower mobility (Fig. 4, bands a, b and d). After a short exposure (6 rain) to trypsin, a new band of higher mobility (band e), intermediate between those of carbonic anhydrase (M, 30,000) and trypsin inhibitor (M, 20,000), was observed. Close inspection of the electrophoretic pattern shows the trace of an additional band (band c) which is also absent in hemoglobin that was not exposed to trypsin. At progressively increasing times of exposure

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to trypsin, the intensity of the high mobility band (band e) increases and that of the other bands decreases. Concomitantly, the bands become more and more diffuse. For the longest exposure shown, the electrophoretic pattern consists of essentially one diffuse band, the one with the high mobility observed starting from very early stages of trypsinolysis. Similar results were obtained by limited proteolysis with subtilisin. The three elcctrophorefic bands common to hemoglobin that had not been exposed to trypsin and hemoglobin that has been exposed to it for short times were identified, in accordance with a previous study (Darawshe et al., 1987), as individual, and associations of two and three, polypeptide chains. On the basis of their mobilities, the two bands appearing upon trypsinolysis were associated with species of half and three halves the molecular mass of a polypeptide chain (Fig. 5). Peptide mapping

Reduced and carboxymethylated Ascaris hemoglobin was subjected to tryptic digestion. A typical peptide map is presented in Fig. 6. The origin shows only slight fluorescence, suggesting that the digestion was virtually complete. It may be noted that a number of spots (six) did not migrate in the electric field. This feature, which reflects the presence of fragments carrying no net charge at the pH of the experiment, has been encountered before (Boeker et al., 1969). The map shown in Fig. 6 contains 29 strong fluorescamine positive spots. Several peptide maps from different digests had 27-30 strong spots. Maps of protein that had undergone digestion for different periods of time (12, 16 and 26 hr) showed a similar pattern.

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Fig. 4. SDS--polyacrylamidegel electrophoresis of Ascaris suum hemoglobin following mild trypsinolysis. Left: Electrophoretic patterns of protein markers (gel 1), hemoglobin that had not been treated by trypsin (2), and hemoglobin exposed to trypsin for 6(3), 15(4), 20(5), 90(6), 150(7) and 330(8) min. a-e denote electrophoretic bands on gels. Right: Densitometric scans of gel 2 (upper scan) and gel 3 (lower scan). The direction of migration is indicated by a horizontal arrow. The positions of the peaks in the lower scan are indicated by vertical arrows.

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Fig. 5. Identification of products generated by mild trypsinolysis of Ascaris suum hemoglobin. Semilogarithmic plot of the molecular mass associated with a given band, M, scaled relative to the molecular mass of a single polypeptide chain, M c, versus band mobility, u. Linearity of the plot was achieved by setting for the electrophoretic bands a, b, c, d and e (Fig. 4) values M/M¢ = 3, 2, 3/2, I and 1/2, respectively. DISCUSSION

The results of this work indicate that hemesaturated Ascaris hemoglobin binds oxygen reversibly. Comparison of the oxy derivative spectrum of this hemoglobin (Fig. 1) with that of hemoglobin isolated from hemolymph where the berne binding sites are only partially occupied (Darawshe et al., 1987, Fig. 1) shows that the maxima of absorption are located, within experimental error, at the same wavelengths. It may therefore be concluded that the extraneous heme added to obtain saturation functions as well as the heme already occupying the partially saturated heme sites. This indicates that the polypcptide chain in Ascaris hemoglobin carries two functional sites for binding oxygen. Ascaris hemo-

globin is similar in this respect to hemoglobins from some crustacea where the molecules are composed of polypeptide chains carrying two heme sites each (Ilan and Daniel, 1979). It has been shown in this work that mild trypsinolysis of Ascaris hemoglobin by trypsin leads to the almost exclusive production of a species half the size of a polypeptide chain. This fact suggests that the cleavage takes place in an exposed segment located midway between the two ends of the polypeptide chain. The protection of the rest of the molecule against proteolytic cleavage is, in all probability, due to folding. The picture that emerges is that of two folded structural units of roughly equal size joined together by the continuity of the polypeptide chain. The major band obtained by the limited trypsinolysis thus corresponds to a single unit. Like the minor bands in the SDS-polyacrylamide gel electrophoretic pattern of Ascaris hemoglobin (Fig. 4, gel (b)) which represent associations of two or more polypeptide chains that persist in the presence of detergent, the minor band in the pattern of hemoglobin that has been exposed to trypsin (Fig. 4, gel (c)) may be explained by the association of an intact polypeptide chain with the product of chains that had undergone cleavage. In this way, the presence of a band of M, three halves that of a polypeptide chain can be rationalized. The results obtained from the peptide mapping of tryptic hydrolysates of Ascaris hemoglobin provide an indirect indication for the occurrence of a repetition in the amino acid sequence. It is known from amino acid composition that the hemoglobin contains 30 lysyl and 22 arginyl residues per polypeptide chain of M r 41,600 (Darawshe et al., 1987). Accordingly, we should anticipate 53 tryptic peptides if

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+ Fig. 6. Peptide map of Ascaris suurn hemoglobin following tryptic digestion. Hemoglobin digests were subjected to electrophoresis in the first dimension followed by chromatography in the second direction, indicated by the arrow. Left: Photograph of TLC plate sprayed by fluorescamine. In this map, 29 strong spots, shown on tracing on the Right, were counted. A small number of weak spots which appeared in some maps but were absent in others, were not included in the count. In the tracing, the location of the origin is indicated by an asterisk.

Structure of Ascaris hemoglobin the polypeptide chain consists of a non-recurring sequence and 27 if it contains a sequence that is once repeated (Klotz et al., 1975). The number of fragments obtained from peptide mapping is in good agreement with that expected if the polypeptide chains are identical and composed of a twice occurring sequence. The findings of this study afford a basis for a better understanding of the structure of the polypeptide chain of Ascaris hemoglobin. The existence of a repeated sequence would explain the folding of the polypeptide chain into two structural units of equal size. The presence of two heme binding sites per polypeptide chain would also be rationalized. Our conclusions are in line with current ideas regarding the multidomain structure of many extracellular invertebrate hemoglobins (Bonaventura and Bonaventura, 1983). Definite confirmation of the occurrence of repeated sequences must await the results of primary structure determinations. REFERENCES

Allen G. (1981) Specific cleavage of the protein. In Laboratory Techniques in Biochemistry and Molecular Biology (Edited by Work T. S. and Burdon R. H.), Vol. 9, pp. 43-134. Elsevier, Amsterdam. Ascoli F., Rossi Fanelli M. R. and Antonini E. (1981) Preparation and properties of apohemoglobin and reconstituted hemoglobins. In Methods in Enzymology (Edited by Antonini E., Rossi-Bernardi L. and Chiancone E.), Vol. 76, pp. 72-87. Academic Press, London. Boeker E. A., Fischer E. H. and Snell E. E. (1969) Arginine decarboxylase from Escherichia coli. J. biol. Chem. 244, 5239-5245. Bonaventura C. and Bonaventura J. (1983) Respiratory pigments: structure and function. In The Molluscs (Edited by Hochachka P. W.), Vol. 2, pp. 1-50. Academic Press, London. Darawshe S., Tsafadyah, Y. and Daniel E. 0987) Quaternary structure of erythrocruorin from the nematode Ascaris suum. Biochem. J. 242, 689~594.

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Di Iorio E. E. (1981) Preparation of derivatives of ferrous and ferric hemoglobin. In Methods in Enzymology (Edited by Antonini E., Rossi-Bernardi L. and Chiancone E.), Vol. 76, pp. 57-72. Academic Press, London. Gracy R., W. (1977) Two-dimensional thin-layer methods. In Methods in Enzymology (Edited by Hits C. H. W. and Timasheff S. N.), Vol. 47, pp. 195-204. Academic Press, London. Huang T.-S. and DeLange R. J. (1971) Egg white avidin. J. biol. Chem. 246, 686-697. Ilan E. and Daniel E. (1979) Structural diversity of arthropod extracellular haemoglobins. Comp. Biochem. Physiol. 6315, 303-308. Ilan E. and Daniel E. 0989) Oxygen binding properties of erythrocruorin from the clam shrimp Caenestheria inopinata. Comp. Biochem. Physiol. 94A, 505-508. Klotz I. M., Darnall D. W. and Langerman N. R. (1975) Quaternary structure of proteins. In The Proteins (Edited by Neurath H. and Hill R.), 3rd Edn, Vol. 1, pp. 293411. Academic Press, London. Lee D. L. and Smith M. H. (1965) Hemoglobins of parasitic animals. Expl. Parasit. 16, 392424. Metzler D. E., Harris C., Yang I.-Y., Siano D. and Thomson J. A. (1972) Band-shape analysis and display of fine structure in protein spectra: A new approach to perturbation spectroscopy. Biochem. biophys. Res. Commun. 46, 1588-1597. Okazaki T., Briehl R. W., Wittenberg J. B. and Wittenberg B. A. (1965) The hemoglobin of Ascaris perienteric fluid: II. Molecular weight and subunits. Biochem. biophys. Acta 111,496-502. Okazaki T. and Wittenberg J. B. (1965) The hemoglobin of Ascaris perienteric fluid: III. Equilibria with oxygen and carbon monoxide. Biochim. biophys. Aeta 111, 503-511. Teale F. W. J. (1959) Cleavage of the haem-protein link by acid methylketone. Biochim. biophys. Acta 35, 543. Weber K., Pringle J. R. and Osborn M. (1972) Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. In Methods in Enzymology (Edited by Hirs C. H. W. and Timasheff S. N.), Vol 26, pp. 3-27. Wittenberg B. A., Okazaki T. and Wittenberg J. B. (1965) The hemoglobin of Ascaris perienteric fluid: I. Purification and spectra. Biochem. biophys. Acta 111, 485495.