THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 246, No. 23, Issue of December 10, PP. 7318-7327, 1971

Pf+-dea in U.S. A.

The Hemoglobin of Arenicola cristata*

(Received for publication, June 21, 1971)

LLOYD WAXMAN$

From the Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

SUMMARY

The chemical and physical properties of the hemoglobin of the polychaete annelid Arenicola cristata have been studied. Gel filtration and ultracentrifugation indicate that it has a molecular weight of 2.85 x lo6 and is composed of 12 subunits of molecular weight 230,000. The iron content corresponds to the presence of 1 mole of heme per 26,000 g of protein. Acrylamide gel electrophoresis in sodium dodecyl sulfate indicates that there are two polypeptide chains with masses of 13,000 and 14,000 daltons. These data suggest that two chains are involved in binding one heme and that there are approximately 96 hemes per molecule. Amino acid analyses of the molecule and its individual chains have been per- formed revealing the presence of three disultide groups and one free sulfhydryl group per heme unit. Oxygenation studies show that the molecule is highly cooperative with a Hill coefficient close to six, but it has a small Bohr effect.

In order to understand the role of proteins in the mediation of cooperativity or information transfer in extended biological structures such as electrical conduction in nerve membranes, I have chosen to study as a model of this phenomenon the proper- ties of annelid hemoglobin, a large, cooperative molecule. Al- though the oxygen-binding properties of this class of proteins have been known for over 40 years and rather extensive electron microscopy of various species has been carried out as well, no detailed structural studies have been made in order to correlate the remarkable functional properties which characterize these molecules with the chemical properties and spatial orientation of their subunits.

Toward this end, I have examined the hemoglobin of the lug- worm Arenicola cristata, whose unusual oxygen-binding proper- ties, first observed by Allen and Wyman (I), single it out as the most cooperative protein molecule reported to date. Clearly, by understanding how such a molecule is organized at its most fun- damental level, one should be able to build a model for the native species which is both consistent with physical studies and will explain the high degree of cooperativity found in many similar molecules.

* This work was supported by Research Grant GB-17953 from the National Science Foundation to Guido Guidotti.

$ United States Public Health Service Trainee (Grant GMO0782) in the Division of Biophysics, Harvard University.

MATERIALS

Sepharose and Sephadex gels and blue dextran were obtained from Pharmacia. Carboxymethylcellulose was purchased from Reeve Angel Company, Clifton, New Jersey, and prepared ac- cording to the manufacturer’s suggestions. Acrylamide, meth- ylene bisacrylamide, and N , N, N’ , N’-tetramethylethylenedi- amine used in all gel electrophoresis experiments were Eastman products. Ampholytes, pH range 4 to 6, were obtained from LKB Instruments (Stockholm, Sweden), and sodium dodecyl sulfate from Sigma.

Marker proteins used in gel electrophoresis experiments and to calibrate Sephadex columns were obtained from Sigma, Mann, and Worthington. Aspartate transcarbamylase from Escherichia coli was the gift of Dr. Jurg Rosenbusch. The virus particles, R17, Q/3, and trit,iated 4X174, used to calibrate Sepharose col- umns, were the generous gifts of Mr. Alan Weiner, Dr. Robert Kamen, and Dr. David Dressler.

N-Ethylmorpholine was redistilled over ninhydrin. N-Ethyl- maleimide was obtained from Mann, and iodoacetamide, 4,4’- dithiopyridine, and p-hydroxymercuribenzoate were Aldrich products. Guanidine hydrochloride was prepared from the carbonate (Eastman) according to the procedure of Nozaki and Tanford (2). Succinic anhydride was also an Eastman product.

Carboxypeptidase A and B, treated with diisopropyl phospho- fluoridate was obtained from Worthington. Polyamide layers (Cheng Chin Trading Company, Taipei, Taiwan) used in sepa- rating dansyll amino acids were obtained through Gallard- Schlesinger Chemical Manufacturing Company, Long Island.

EXPERIMENTAL PROCEDURE

Methods of Pur$cation of Hemoglobin-Hemoglobin was ex- tracted from live worms obtained from the Marine Biological Laboratory, Woods Hole, Massachusetts, essentially as de- scribed by Allen and Wyman (1). The animals were bled im- mediately upon arrival, usually 1 to 2 days after their collection at Woods Hole, to ensure that the hemoglobin would not begin to decompose as the animals died. In order to obtain the blood, in which the pigment occurs free in solution, the animals were first rinsed in distilled water and then put in a solution of 0.5 M

MgSOc for several minutes in order to partially anesthetize them. They were then pinned to a block of Styrofoam and the body wall incised with a razor blade, exposing the dorsal blood vessel. Body fluid was sponged up as well as possible to avoid contami- nation by lipids and other proteins as well as dilution of the blood.

1 The abbreviations used are: dansyl, 5-dimethylaminonaph- thalene-l-sulfonyl; SDS, sodium dodecyl sulfate; NEM, N-ethyl- maleimide.

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A 2-ml hypodermic syringe equipped with a l-inch number 24 needle was inserted into the blood vessel and the blood removed. In this way, 0.1 to 0.4 ml of a 6 to 8% solution of hemoglobin was collected per animal. In larger worms, some of the smaller blood vessels were also used, but any blood which inadvertently leaked out was not collected.

The solution collected from a dozen worms (usually about 2 ml) was centrifuged at 4” for 20 min at 8000 X g to remove any particulate matter which may have been collected. The solution was then chromatographed on a Sepharose 2B column, 2.3 X 38 cm, at 4” in 0.05 M Tris-hydrochloride, 0.2 M KCl, 1 mM EDTA, pH 7.2. The fractions containing hemoglobin were pooled, con- centrated by vacuum dialysis, and used immediately for oxy- genation experiments or else frozen for future use in structural studies.

Oxygenation-The binding of oxygen was determined accord- ing to Benesch, Macduff, and Benesch (3) and the spectra re- corded on a Gary 15 spectrophotometer. A small amount of the purified concentrated protein or unpurified hemoglobin was di- luted in a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, ionic strength 6.1, at various pH values. Con- centrations were estimated assuming a molar extinction coeffi- cient at 540 nm for oxyhemoglobin of 1.5 X lo4 cm-l M-I per heme (3).

Ultracentrifugation-Sedimentation velocity studies on the purified hemoglobin or its breakdown products were performed in a Beckman model E analytical centrifuge at 20” with a double sector cell.

Diffusion coefficients were determined according to Kawahara (4) in a synthetic boundary cell after exhaustive dialysis against 0.05 M Tris-HCl, 1 mM EDTA, pH 7.2. Photographs of the schlieren peaks were magnified 10 times and the areas measured with a planimeter.

The partial specific volume was calculated from the amino acid composition according to the method outlined by Cohn and Edsall (5), and the density of the 0.05 M Tris buffer used in most ultracentrifuge experiments was determined pycnometrically in a Gay-Lussac specific gravity vessel.

G’el Filtration-An approximate size for the intact hemoglobin molecule was obtained by gel filtration on a Sepharose 2B column, volume 415 ml, at 4” in 0.1 M sodium phosphate, 0.2 M KCI, pH 7.0. The column and void volumes were determined by eluting potassium ferricyanide and blue dextran. The column was then calibrated with the virus particles R17, Qp, and triti- ated 4X174. In addition, hemoglobin from the earthworm Lumbricus terrestris was chromatographed on a 170-ml Sepharose 2B column which had been used for the purification of Arenicola hemoglobin. The virus particles were detected spectrophoto- metrically by their absorbance at 260 nm, except 4X174, which was assayed by radioactive counting. R17 was used as an internal standard in all experiments. I The size of the subunit formed upon incubation at pH 10.4 was determined on a 440-ml G-200 column in the same buffer as the Sepharose column. The void and column volumes were determined as described above, and the column was calibrated with aspartate transcarbamylase from E. coli, rabbit muscle aldolase, and bovine catalase. Elution position was determined by optical density readings at 230 nm, and aspartate trans- carbamylase was used as an internal standard in all experi- ments. Data were analyzed according to Ackers (6) or by plot- ting the distribution coefficient, Kd, versus log molecular weight.

Production of Subunits-As shown by Svedberg and Eriksson- Quensel (7), annelid hemoglobins dissociate at high pH. To make use of this property, the hemoglobin was dialyzed for 12 hours at 4” into a sodium hydroxide-glycine buffer, 1 mM EDTA, ionic strength 0.1, at pH 10.4. By centrifugation, the product consisted primarily of a 10.3 S particle. Dialysis at 4” against 0.1 M phosphate, 0.2 M KCl, 1 mM EDTA buffer, pH 7.0, yielded products which eluted in the void volume of the calibrated G-200 column as well as giving an elution pattern characteristic of an associating system. Consequently, the column was re-equili- brated in the dissociating pH 10.4 buffer, and aspartate trans- carbamylase run to ensure that the parameters of the column had not changed.

Disc Electrophoresis-To determine the molecular weights of some of the possible subunit complexes, the hemoglobin was dialyzed at pH 10.4 against the sodium hydroxide-glycine buffer at 4”, then against 0.06 M imidazole-hydrochloride, pH 5.7, and run on polyacrylamide gels in an asparagine-Tris buffer, pH 7.3, as described by Hedrick, and Smith (8). (By means of their method for analyzing mobility data, it was possible to determine a subunit molecular weight.)

The isoelectric point of the native molecule was determined by isoelectric focusing, following the method of Wrigley (9), on ampholyte gels which were 5% in acrylamide. The gels were focused for 5 to 7 hours at 4”, cut into l-cm pieces, rinsed with deionized water, and each piece put into a separate flask eon- taining 4 ml of water. The pieces were shaken overnight at room temperature, and the pH of the solution in each flask meas- ured on a pH meter. The position of the hemoglobin band on a plot of distance against pH gives the isoelectric point directly.

Gel electrophoresis in SDS was performed according to the methods described by Shapiro, Vmuela, and Maize1 (10) and Weber and Osborn (11). Protein samples were first boiled for 3 min in a solution 1 y. in SDS and 2-mercaptoethanol to eliminate the possibility of proteolytic degradation known to occur in SDS, as shown by Pringle (12). The samples were then dialyzed against a solution which was 0.1 y. in SDS and 2-mercaptoetha- nol, 0.01 M in sodium phosphate at pH 7.1. Marker proteins were prepared in the same way.

In those experiments where NEM replaced 2-mercaptoethanol, the concentration of NEM was maintained at 1 To. A variation of this procedure consisted of staining one gel to determine the position of the protein, cutting out bands from unstained gels which had been frozen, eluting overnight in 0.1% SDS, and re- running the protein. Gels were scanned on the modified Hitachi- Perkin-Elmer 139 spectrophotometer of Berg (13).

Iron Determination-The heme concentration was estimated spectrophotometrically after converting purified hemoglobin to the cyanmet derivative. The molar extinction coefficient of this derivative is 11.5 X lo4 M-’ cm-l at 540 nm (14). These results were confirmed by atomic absorption spectroscopy with the use of a Jarell-Ash apparatus. A standard calibration curve was made from a series of solutions of different concentrations of ferrous ammonium sulfate which had been dissolved in 0.1%

HCl. The amount of protein in the sample was determined from a

24-hour quantitative amino acid analysis with norleucine as an internal standard. The values obtained for aspartic acid, glu- tamic acid, alanine, valine, leucine, and phenylalanine were prorated relative to the amounts found in the native molecule.

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Thus, the total amount of protein in the sample relative to the amount of iron in the same sample could be ascertained.

Amino Acid Analyses-The amino acid composition was found by hydrolyzing samples of purified hemoglobin for 24, 48, and 72 hours in 6 N HCl at 110” in evacuated glass tubes with nor- leucine as an internal standard. A Beckman-Spinco amino acid analyzer model 120C with the chromatographic system of Spack- man, Stein, and Moore (15) was used. Tryptophan was esti- mated spectrophotometrically with acid-acetone-extracted globin by the method of Edelhoch (16) and methionine and half-cystine as their performic acid derivatives (17). Color constants were determined by derivatizing the free amino acid or glutathione and found to agree with those reported in the literature. The presence of sugar was assayed by the anthrone reaction (18) with the use of glucose as a standard and by staining SDS polyacryla- mide gels for glycoprotein with fuchsin, as described by Zacha- rius et al. (19).

Chain Separation-Globin was prepared by adding 10 ml of cold acid-acetone (acetone, 500 ml; concentrated HCl, 10 ml; and glacial acetic acid, 25 ml) to 2-ml samples of salt-free solu- tions of hemoglobin (20). The precipitated globin was washed several times with 10 ml of the cold acid-acetone and once with cold acetone. Two to 10 mg were carboxymethylated with iodo- acetamide according to Crestfield, Moore, and Stein (21) in 6 ml of 6 M guanidine hydrochloride.

The reaction was allowed to proceed for 30 min at pH 8.0, stopped by the addition of 2 ml of glacial acetic acid, and the solution dialyzed against 0.2 N acetic acid for 24 hours and then against water for 48 hours with repeated changes. The protein solution was then concentrated by vacuum dialysis to 1 ml and dialyzed against a solution of 6 M urea, 5 mM sodium citrate at pH 5.9. The dialysate was then applied to an 8ml carboxy- methylcellulose column in the same solvent. After 20 ml of eluent had been collected a linear salt gradient was begun, con- sisting of 200 ml each of starting buffer and 0.2 M citrate. Al- ternatively, the globin was not alkylated, but reduced in 0.5% 2-mercaptoethanol which was then put in all buffers.

Protein was monitored by absorbance at 280 nm and the peaks characterized by SDS gel electrophoresis following dialysis to eliminate salts and urea. Since each peak was found to consist of both a high and low molecular weight species, a second separa- tion was performed by applying the concentrated peaks to a G-200 column in 1% SDS, pH 8.5, 0.05 M borate.

Sulfhydryl Determination-Because of the strong absorbance of heme at most wave lengths where spectrophotometric sulfhy- dry1 assays are performed, the free sulfhydryl content was deter- mined by titrating with 4,4’-dithiopyridine and reading the change in absorbance at 328 nm, as discussed by Grassetti and Murray (22). To check the validity of the method when applied to heme proteins, human hemoglobin was used, and the assay carried out both in phosphate buffer and in 6 M guanidine hydro- chloride. Values of 1.4 and 1.6 free sulfhydryls per heme were found for human hemoglobin in guanidine, which compares very well with the accepted value of 1.5.

Carboxymethylation with iodoacetamide, as discussed above, was carried out in the presence and absence of 2-mercaptoethanol and the protein analyzed for carboxymethylcysteine. Similarly, the native protein was added to a 3% solution of SDS, 0.01 M

sodium phosphate, at pH 7.1, which was 1% in NEM. The reaction was permitted to run for 1 and 5 hours and the suc- cinylcysteine produced was analyzed according to Smyth, Blu-

menfeld, and Konigsberg (23). In both experiments, human hemoglobin was used as a check.

Amino- and Carboxy-terminal Amino Acid Determinations- By means of unmodified, performic acid-oxidized, or reduced and carboxymethylated protein, amino-terminal determinations were made following the cyanate procedure of Stark and Smyth (24). The method was modified by scaling down all steps by a factor of 10. The system was tested with human hemoglobin and yielded 0.8 mole of valine per 15,000 g of protein.

To corroborate the cyanate procedure, the dansyl method was employed, as described by Hartley (25). The dansyl derivatives were identified by three dimensional thin layer chromatography on polyamide plates (5 x 5 cm) which are coated on both sides so as to permit simultaneous chromatographing of unknown and standard dansylated amino acids (26). In this way, one could assign the correct NHz-terminal amino acid to its polypeptide chain by dansylating protein samples of the separated chains.

The correspondence between these results and the bands found on SDS gels of the total protein was made in the following way. Hemoglobin was dansylated in the usual manner, dissolved in 1 To SDS, 1 To 2-mercaptoethanol, usually with heating, dialyzed against SDS gel buffer overnight, and run on 10% acrylamide SDS gels. The bands were detected with ultraviolet light, cut out, and hydrolyzed for 24 hours in 6 N HCl. The hydrolysate was then centrifuged to pellet the acrylamide, and the super- natant brought to dryness on a flash evaporator. End group determinations were then performed as described above. The COOH termini were investigated with carboxypeptidase A and 23, prepared from the commercial enzymes according to Ambler (27). Digestions were carried out at 26”, pH 7.65, in a 0.2 M

sodium phosphate buffer containing 0.056 M SDS, as described by Guidotti (28), with an enzyme to substrate ratio of 1:40. Aliquots were removed at time intervals and the reaction stopped by 1 N HCl. The amino acids present in the supernatant were determined directly on the amino acid analyzer following cen- trifugation of the precipitated protein.

Eflects of Salts-To help form a better idea of the relative strengths of the bonds holding the subunits together, and in doing so, to characterize the unit of maximum cooperativity, I examined the influence of various salts on the integrity of the native molecule. In addition to studying the effect of glycine buffer at pH 10.4, I examined 2 M and 6 M urea, 6 M NaCl, 0.4 M

and 1 M MgC12, all at pH 7.2, and 0.2 M NaH2POk at pH 10.4. In each case, a small amount of concentrated hemoglobin was dissolved in the solvent of interest and centrifuged immediately, or else dialyzed against the solvent for varying periods of time at 4”. In addition, attempts were made to reconstitute the molecule by dialysis at neutral pH and 4” in phosphate buffer. Density and viscosity corrections were made from values given in the International Critical Tables (29) and by Kawahara and Tanford (30).

Chemical Modi$cation-Attempts to disrupt the molecule by reacting the free sulfhydryl groups were made with NEM, 4,4’-dithiopyridine, and p-hydroxymercuribenzoate. Solutions which were 0.3% hemoglobin, 0.1 M sodium phosphate at pH 7, and 1 y. in one of the above reagents were incubated for 1 hour at room temperature and then run in the ultracentrifuge.

Succinylation was also used as a more severe means of modifi- cation. Samples of the protein (2 to 10 mg) were reacted with succinic anhydride according to Klotz (31). The reaction was carried out at pH 8.0 in 0.1 M Tris-HCl at 0”. The amount of

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. ($ 46

42-

36- . I I I

02 06 1.0 1.4 16

mg Protein/ml

FIG. 1. Dependence of the sedimentation constant upon the concentration of Arenicola hemoglobin. All experiments carried out at 20” in 0.05 M Tris-HCl, 1 mM EDTA, pH 7.2.

anhydride added, usually over a period of 1 hour, was 5 and 10 times the number of moles of lysine calculated to be present in the reaction solution. The pH of the reaction was monitored with a p1-I meter equipped with a microelectrode and maintained by the addition of 0.2 M Na2C03. Dialysis was carried out at 4” against 0.5 M NaCl, 0.01 M phosphate, pH 7.2, and sedimentation velocity experiments were performed on the dialyzed solutions.

Acetylation experiments carried out with acetic anhydride in 0.2 M phosphate buffer at pH 7.5 and 0” according to Bucci et al. (32) gave similar results and will not be discussed here.

RESULTS

PurQication of Henloglobin-Filtration of the hemoglobin on Sepharose 2B showed that there was no other protein component associa,ted with the blood, at least from the criterion of absorb- ance at 280 nm. The presence of only one peak upon ultra- centrifugation and the clean banding pattern found on SDS gels corroborate the effectiveness of this step.

Physical Parameters of Molecule-The sedimentation constant was determined at a series of concentrations and extrapolated to zero concentration to give a value of szo = 56.2 S (Fig. 1). Cor- rection for the density of 0.05 M Tris at 20”, which was found to be 1.009 g per cc (average of three determinations) gave sZO,w = 57.8 s.

Determination of the diffusion coefficient at 0.5%, 0.6%, and 1.45yo gave values of D, = 1.855, 1.80, and 1.85 X lo-’ cm2 per set, respectively, thus indicating little concentration dependence.

The partial specific volume determined from the amino acid composition was found to be 0.726 cc per g. By means of the Svedberg equation, this gives an approximate molecular weight of 2.85 x 106.

When the protein was chromatographed on a Sepharose 2B column calibrated with several roughly spherical virus particles, it was found to elute with R17 (Table I). With the use of the best data in the literature, analysis by the method of Ackers (6) gives a Stokes radius of 132.5 A. The Einstein relation, in conjunction with the Stokes radius, gives a derived D20,w = 1..62 x low7 cm2 per sec. Rrenicola hemoglobin is not a spherical particle, however, but in fact is closer in shape to a torus (Fig. 2). Consequently, the fact that the structure contains voids makes the relation between size and elution position different from that of the spherical viruses used t’o calibrate the column.

TABLE I

Gel jiltration of intact hemoglobin molecule

The size of Arenicola hemoglobin was estimated by gel filtration on a Sepharose 2B column calibrated with several virus particles, and the data analyzed according to Ackers (6). Experimentally and derived column parameters: VO = 240 ml, Vi = 415 ml, a0 = 55.4 A, bo = 56.8 A.

Particle /

V, /

Ki Diameter D X 107

(-4) cm2 per set

ml

R17 (33)” . 311.5 0.172 266 Q/3 (34)0.... ._.. 312.0 0.172 250 1.55 +x174 (35)a. . . . . 306.1 0.159 271 f 13.5 Arenicola. 312.5 0.174

a The number in parentheses is a reference.

FIG. 2. Electron micrograph of native Arenicola hemoglobin. Negative staining with uranyl acetate. X 450,000.

Kd FIG. 3. Gel filtration of Arenicola subunit on Sephadex G-200

column, equilibrated with 0.1 M phosphate, 0.2 M KCl, pH 7.0. The flow rate was 8 ml per hour at 4”. Fractions (3 ml) were collected and monitored at 280 nm. The markers used were A, aspartate transcarbamylase, B, bovine catalase, and C, rabbit muscle aldolase. Kd is the partition coefficient; V0 = 440 ml, V, = 121 ml; aa = 21.7 A, bo = 26.4 A.

Interestingly, both Arenicola and Lumbricus hemoglobin were found to elute within 1 ml of each other on a smaller Sepharose column, Vi = 170 ml, which was used for purification purposes. Lumbricus hemoglobin has an experimentally determined D20,w = 1.81 x 1OP cm2 per set (36).

Determination of Subunit Molecular Weight-In studying the

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TABLE II

Summary of results sf yeljltration experiment on Arenicola subunit

Method of analysis Dzo, to X 10’ cm2 per set Molecular weight

Graphical Ackers (6)

g/mole

4.2 (not shown) 2.2 X IO5 (Fig. 3) 4.24 (a = 50.6 A) 2.28 x 105

I I I 2 6 IO 14

Gel concentration %

L I

20 B I I I I

I I I I I 0 5

I J 15 25 35

Molecular weight Ix 10m4)

FIG. 4. Determination of the subunit size of Arenicola hemo- globin by polyacrylamide gel electrophoresis analyzed according to Hedrick and Smith (8). A shows the effect of different acryl- amide concentrations on mobility for the five major bands. The negative slopes of the lines obtained from such a plot are noted. B is the slope-molecular weight relation of the standard proteins : A, horse liver alcohol dehydrogenase; B, bovine serum albumin; C, horse liver alcohol dehydrogenase dimer; D, bovine serum albumin dimer; E, yeast glyceraldehyde 3-phosphate dehydro- genase; F, yeast alcohol dehydrogenase; G, bovine serum albumin trimer; H, bovine catalase; I, aspartate transcarbamylase. The arrows indicate the Arenicola bands, corresponding to molecular weights 53,000 to 58,000 and 232,500. R, is the mobility with respect to marker dye.

effect of ~1-1 on the sedimentation of giant hemoglobins, Svedberg and Eriksson-Quensel (7) noted that at values greater than 8, the molecules begin to break down into smaller particles. Levin (37) has also given evidence for this in electron microscopy studies of molecules which were subjected to high pH conditions. Incubation at pH 10.4 in a sodium hydroxide-glycine buffer resulted in the production of several sizes of particles, approxi- mately one-quarter of which eluted in the void volume of a G-200 column, while the remainder was divided between a large peak

FIG. 5. Electrophoretic patterns of Arenicola hemoglobin on 10% acrylamide gels in 0.1% SIX From left to right: A, pattern obtained with the reduced protein showing the presence of mono- mers and dimers; B, pattern obtained after incubation of the protein in 1% NEM; C and D, patterns obtained with the first and second peak, respectively, from CM-cellulose chromatog- raphy; E and F, electrophoresis of the components in Peak C after their separation on G-200 in 1% SDS; G and H, electrophore- sis of the components in Pea/c D after their separation on G-200 in 1% SDS. See under “Experimental Procedure” for details.

eluting in the column volume and a smaller peak eluting near the front of t.he column. These experiments point to a subunit molecular weight of 2.2 x IO” to 2.3 x lo5 by several methods of analysis (Fig. 3, Table II).

To corroborate this result’, the native protein was first dialyzed against the same high pH bufler for 6 hours at 4”, and then against an application buffer consisting of 0.06 M imidazole-hy- drochloride, pH 5.9. Gel electrophoresis according to the method of Hedrick and Smith (8) gave a particle weight of 232,500 g per mole, and in addition, a smaller subunit value of 5.3 to 5.5 x lo4 g per mole was found (Fig. 4B). The parallel lines in t,he plot of log mobility against acrylamide concentration (Fig. 4A) may be interpreted as being due to charge isomers, and those which intersect as size isomers (8). This suggests that the smaller particle is derived from the larger.

Molecular Weights of Polypeptide Chains-SDS gel electro- phoresis always gives the banding pattern shown in Fig. 5A. Reduction and carboxymethylation decreases the intensity of the upper two bands, but it is difficult to eliminate them com- pletely. Integration of the scans of these gels at several wave lengths between 500 and 600 nm shows that approximately 90% of the total protein is found in the two lower bands, and that the sum of the number of moles of protein in the lower band of each pair is equal to the sum of the moles in the upper bands in each pair. Calculation of the molecular weights according to Weber and Osborn (11) suggests that the upper pair are dimers of the lower pair (Fig. 6). The existence of dimers is not unusual, as higher aggregates have been found in many proteins which contain disulfides, such as y-globulin. The values for these molecular weights, although highly reproducible, are subject’ to certain qualifications, as shown by Fish, Reynolds, and Tanford (38); below molecular weights of 15,000, the relation between mobility and molecular weight is no longer reliable or predictable,

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I I I I I , I -1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Mobility

FIG. 6. Determination of the molecular weights of the individ- ual polypeptide chains of Awnicola hemoglobin. The marker proteins used in this experiment were A, bovine glutamic dehy- drogenase; B, pig heart, fumarase: C, yeast alcohol dehydrogenase; D, C chain of aspartate transcarbamylase; E, horse heart cyto- chrome c dimer; F sperm whale myoglobin; G, R chain of aspartate transcarbamylase; H, ribonuclease; I, cytochrome c. All proteins were run on duplicate gels. The arrows indicate the mobilities of the Are&cola subunits. The average molecular weights for 75 gels are 13,000, 14,250, 26,000, and 28,000.

and the hydrodynamic size of an unreduced protein is smaller than that of the reduced protein.

Omission of an incubation or dialysis step in preparing samples for SDS gel electrophoresis increases the intensity of the upper bands at the expense of the lower, but in addition, a faint band appears at molecular weight 54,000. Incubation in SDS with 1 y0 NEM instead of 2-mercaptoethanol gives the pattern shown in Fig. 5B. At an NEM concentration of O.l%, small amounts of the lower bands are detectable as well. Elution of the protein from the high molecular weight band of unstained gels, followed by dansylation to make detection more sensitive and rerunning after incubation in 1% 2-mercaptoethanol, reveals two faint low molecular weight bands and no others.

Separation of Chains-A characteristic of annelid hemoglobins is their low isoelectric point. When run on 5% acrylamide, pH 4 to 6 ampholyte gels, Lumbricus hemoglobin was found to have a p1 of 5.15, which compares very well with the value of 5.28 given by Svedberg and Pedersen (36). Arenicota hemoglobin has a p1 of 4.5 to 4.6, which is identical with the value found for Arenicola marina, 4.56 (36).

Because the isoelectric point is so low, chain separation was attempted on carboxymethylcellulose at pH 5.9. The protein generally eluted in two peaks, the first consisting of material which did not adsorb to the column. A typical elution profile is shown in Fig. 7A, and gels corresponding to the protein peaks in Fig. 5, C and D.

When it became clear from SDS gels that each peak consisted primarily of the monomer of one component and the dimer of the other, a separation by size was carried out on a G-200 column in SDS (Fig. 7, B and C). SDS gel electrophoresis of fractions from these peaks revealed the separation of all four components, al- though the lower component from the second peak of the car- boxymethylcellulose column was occasionally contaminated by the other monomer species (Fig. 50). This is probably a result of breakdown of the dimer in this fraction into monomers.

LdO- GRADIENT A-

0.80-

0.60-

.I 0 4 12 20 28 36 44 52 60 68

Elution volume (ml)

Effluent volume (ml) FIG. 7. A, separation of Arenicola polypeptide chains on a

carboxymethylcellulose column, 0.75 X 10 cm. The starting buffer was 5 mM sodium citrate, 6 M urea, pH 5.9. Flow rate was 15 ml per hour, and 2-ml fractions were collected. B and C, sepa- ration by gel filtration of high and low molecular weight species present in the first and second peaks, respective11 , from the sepa- ration on carboxymethylcellulose. Protein samples dialyzed against 1% SDS and 1% 2-mercaptoethanol were applied to a G-200 column, equilibrated with 1% SDS and 0.05 M sodium borate, pH 9.0. The flow rate was 7 ml per hour, and 3.5.ml fractions were collected. Vo = 410 ml, Vi = 140 ml.

Characterization oj Chains-Table III shows the amino acid composition of the intact hemoglobin and of the isolated chains. The chains are designated I and II, and they correspond to the larger and the smaller of the two components, respectively. Both chains contain histidine residues and thus cannot be excluded from heme binding on this basis. However, there are definite differences in the amino acid compositions of chains I and II, the most notable being those in the content of aspartic acid, glutamic acid, and histidine.

The cyanate procedure, while not quantitative, shows that there are two amino-terminal groups (Table IV). The lack of quantitation is probably due to less than optimal recovery of the

amino-terminal amino acids, as reflected in an 80% recovery of

the end group of human hemoglobin. The identification of two NHz-terminal amino acids was sub-

stantiated by end group analysis by dansylation. The dan- sylated protein was separated into its constituent chains on SDS gels, and the protein was obtained as described under “Experimental Procedure.” The results showed that aspartic

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Vol. 246, No. 23

T.\ISLR III

Rc,sulls of amino acid analyses of A. ci~istata and its individual polypeptide chains

Amino acid

I,ysine ................ Histidine ............. Arginine ............ Aspartic acid. ........ Threonine .......... Serine ................ Glutamic acid. ....... Proline. .............. Glycine ............... Alanine ............... Valine ................. Methionine ........... Isoleucine ............ Leucine. .............. Tyrosine. ............. Phenylalanine ........ Tryptophand. ......... Half-cyst,inee. ........ Neutral sugar1 ........

-

residaes/ 27,400 g

12 9

14 30 10 12 28

7 17 22 19 GC 8

21 5

12 5 7 0 OGo/,

Chain I*

residaes/ 14,450 g

5.75 3.4 7.8

16.4 4.4 5.15

16.8 3.2

11.0 12.8 10.5 3.0 4.8 9.95 2.2 6.05 2.05 3.1

-

-

Chain I@

residws/ 13,zoog

6.3 5.6 7.3

13.2 2.8 4.3

10.7 3.55 9.7

10.8 8.6 3.2 3.55

11.0 3.2 5.6 2.8 3.8

lz Results are expressed on the assumption that the two chains arc present in equimolar amounts. Values obtained at three hydrolysis times (see under “Experimental Procedure”) were extrapolated to zero time in the case of threonine, serine, and ty- rosine, and to infinite time in the case of valine. Average values are reported for the remaining amino acids, rounded off to the nearest integer.

* Subunit compositions are the averages of three 24.hour hy- drolyses of different separation experiments.

c Methionine was determined as methionine sulfone. d Tryptophan was determined spectrophotometrically (16). E Half-cystine was determined as cysteic acid in the case of the

whole molecule and in the subunits. f Neutral sugars were assayed by the anthrone reaction (18);

the assay for glycoprotein was negative.

TABLE IV

NHZ-Terminal amino acid determination by cyanate procedure

Results of the cyanate procedure performed under various hy- drolysis conditions according to Stark and Smyth (24). All other amino acids, including cysteic acid, were present in amounts less than 1 mole per 300,000 g of protein.

Amino acid 24 hours in 0.2 96 hours in 48 hours in N NaOH 6NHCI 6ivIICI

Aspartic acid.. 1 male/49,800 g 1 male/49,400 g Glutamic acid. 1 male/50,200 g 1 male/46,300 g Glycine.. 1 male/81,900 g Alanine. 1 male/305,000 g S-Carboxy-

methylcys- teine 1 male/205,000 g

acid is at the NH2 terminus of the larger polypeptide while glutamic acid is the amino end group of t,he smaller chain. The dsnsyl procedure was then carried out on the four fractions which had been isolated by ion exchange chromatography and SDS gel

T.\TsLE v

Amino acids released Jrom A renicola hemoglobin-treated with carboxypeptidase A and IZ

Carboxypeptidase A was added init,ially; carboxypeptidase B was added after 60 min.

Amino acid

Alaninc. Lerrcine Valine.. Isolerlcine Lysi ne Histidine. Argininc

30 min 1 90min 1 120 min / 210 min

lldesl28,000 g polein

0.6X 0.91 1.00 1.00 0.1n 0.38 0.45 0.46 0.16 0.23 0.33 0.37

O.lG 0.24 0.31 0.10 0.25 0.28

0.10 0.26 0.30 0.41 0.25 0.33 0.42

‘l-MILE VI

Resu~‘fs of experiments to determine status of sulfhydryls in Arenicola hemoglobin

Method

Performic oxidation.. Reduction and carboxymethylation in

6 1~ guanidine HCl. Carboxymethylation without reduc-

tion in G M guanidine IICl. Incubation with 1% NEh/L in 3c/, SDS,

pH 7.1 (1 and 5 hours). Incubation with 1% NISM in phos-

phate buffer, pH 7.1 (1 and 5 hours). Reaction with 4,4’-dithiopyridine in 6

M guanidine hydrochloride, pH 7.2 Reaction with 4,4’-dithiopyridine in

phosphate buffer, pH 7.2..

Number of sulfhydryls per27,400g

(3.9

5.8

0.95

0.7

0.7

0.6

0.1

Number of leterminations

3

1

10

2

2

3 2

electrophoresis. The large dimer had aspartic acid at its amino terminus, and the small dimer had glutamic acid. The small monomer also had NHn-terminal glutamic acid, but the band in the position of the large monomer had both end groups. This was probably due to incomplete resolution of the monomer bands in this case.

Treatment of the hemoglobin with carboxypcptidase A-diiso- propgl fluorophosphate released 1 mole of alanine per 28,000 g of protein after a 2-hour digestion. The use of carboxypeptidase B-diisopropyl fluorophosphate, which had considerable carboxy- peptidase A activity, either alone or in combination with carboxy- peptidase A did not alter this result. This suggested that one chain had alanine as its COOH-terminal residue, while the other chain was blocked or perhaps ended in proline, in which cast it would not be attacked by carbosypeptidase A or R. A typical experiment is shown in Table V.

A l-hour digestion with carboxypcptidase A of the larger dimer gave essentially the same amino acid release as the whole mole- cule, while a l-hour digestion of the smaller monomer and smaller dimer showed no significant release of any amino acid.

Iron DetenGnatio?L-By means of two different assays, atomic absorption and spectrophotometric determination following con- version to the cyanmet derivative, iron determinations were made on hemoglobin samples taken from three different preparations of

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Issue of December 10, 1971 L. Waxman

M

1.0 A I

0.8 l---i7

* t/t -1 fn=57 1

7 1

jJ ,I j/f, 1 -0.2 0.2 0.6 1.0 -0.2 0.2 0.6 1.0

Log PO2

FIG. 8. Oxygen equilibrium curve (A) and Hill plot (B) of 0.15% solution of Arenicola hemoglobin. Potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, ionic strength 0.1, pH 7.6, temperature 22”. P = fractional saturation with oxygen.

purified protein; the correspondence in each case was within 5%. The value for the ratio of protein to heme for three experiments was 26,100 i 400 g per mole of heme.

Suljhydryl Determination-Inability to titrate the hemoglobin with p-mercuribenzoate or Ellman’s reagent suggested the pres- ence of disulfide bonds and led to the experiments summarized in Table VI. These results suggest one free sulfhydryl and six involved in disul6de linkages per heme or two polypeptide chains.

Effect of Salts and Chemical Modijkation on Native Hemoglobin -In preliminary experiments to characterize the functional subunit of Arenicola hemoglobin, the effects of several salts and chemical modifications on the sedimentation of the protein in the ultracentrifuge were investigated. These experiments can be briefly summarized by noting that the many procedures used all caused the molecule to break down into varying proportions of two components, one with a sedimentation coefficient of -11 S and the other with one of -5 S. More detailed studies were not undertaken because both high concentrations of salts and succinylation caused the rapid oxidation of the hemoglobin, rendering it unsatisfactory for oxygenation studies.

Incubation with the thiol reagents, NEM, p-mercuribenzoate, or 4,4’-dithiopyridine had no effect on the size of the hemoglobin molecule. Concentrations of NaCl up to 6 M, MgClz up to 1 M,

and urea up to 2 M were also ineffective as dissociating agents, although NaCl and urea caused heme oxidation and 6 M urea brought about aggregation.

Oxygen-binding Properties-Preliminary experiments of the oxygen-binding capability of the hemoglobin confirm the earlier findings of Allen and Wyman (1). Characteristic of the molecule is its asymmetric oxygenation curve, Hill coefficient between 5 and 6, and apparent lack of a Bohr effect (Fig. 8). The free energy of interaction, as defined by Wyman (39), was calculated to be 1800 cal per mole, a value considerably lower than that of mammalian hemoglobins. Some of these results are summarized in Table VII.

TABLE VII Oxygen-binding properties of Arenicola hemoglobin

All experiments were carried out in phosphate buffers, ionic strength 0.1, and at temperatures 22-24”. Hemoglobin concen- trations were 0.1 to 1.2%.

PH Hill PSO Free energy coefficient, n of interaction

mwz Hg cd

5.8 5.4 3.6 1700 7.0 5.0 4.0 1800 7.6 5.7 4.1 1800

DISCUSSION

The data presented in this paper suggest a general structural model for annelid hemoglobins. As summarized by Svedberg and Pedersen (36), the annelid hemoglobins have many proper- ties in common. Their molecular weights are all in the range 2.7 to 3 x 106; their sedimentation constants are from 56 to 61 S; the frictional ratio, f/fo, is usually 1.21 to 1.25; and in all cases, electron microscopy reveals a two-tiered structure consisting of two six-membered rings of diameter 255 to 260 A.

The results presented here indicate that the hemoglobin of A. cristata has a molecular weight of 2.85 x 106. It is composed of two types of polypeptide chains, one with a mass of 13,000 and the other with a mass of 14,000 daltons, present in equimolar amounts. The large polypeptide has NHz-terminal aspartic acid and COOH-terminal alanine. The small chain has NH2- terminal glutamic acid. There is one heme per 26,000 g of pro- tein, which corresponds to one heme per two polypeptide chains, or one heme for the sum of one large and one small polypeptide chain. The studies of the sulfhydryl groups and the results of the SDS gel electrophoresis in the presence of the -SH scavenger NEM indicate that four chains, two of one kind and two of the other kind, are held together in a complex by disulfide bonds. This molecule with a mass of 54,000 daltons would have two hemes, two free -SH groups, and it would be stabilized by six disulfide bonds. The arrangement of the bonds is at present under study. However, the fact that even after very vigorous reduction dimers of identical chains persist indicates that the disulfide bonds between like chains are more stable than those between unlike chains. Whether or not this point has any bear- ing on the way the heme group is held by the chains, indeed the nature of the heme binding site (on one chain or shared by both chains) remains to be described. The many possibilities need not be advanced here.

The molecular weight of another stable subunit of the molecule is 230,000. This subunit is therefore composed of eight poly- peptide chains of each type and it has eight heme groups. This means that this subunit is composed of four of the units with a mass of 54,000 daltons described above and also that it corre- sponds to one-twelfth the mass of the intact molecule (that is, it represents one of the six members of the ring seen by electron microscopy as shown in Fig. 2). This result is incompatible with the notion that the subunit has cyclic a-fold symmetry, as has been suggested for the hemoglobin of Spirographis (40) and of Lumbricus (41), because eight heme groups cannot be dis- tributed symmetrically between the three elements of the sub- unit. However, Levin’s electron micrographs (37) indicate that for this hemoglobin the subunits may have a 4-fold axis of sym-

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7326 Arenicola Hemoglobin Vol. 246, No. 23

metry, or that they are at least rhombus-like. I suggest that the subunit of Arenicola hemoglobin, which contains eight heme groups, has a tetrahedral arrangement of its components, each component having a mass of 54,000 and two heme groups.

Since the intact hemoglobin molecule has 12 subunits of molecular weight 230,000, arranged in two six-membered rings, I envisage that in the rings each subunit (of mass 230,000) is bonded by heterologous bonds to two other subunits, and that

six such subunits form a ring. The ring therefore has cyclic 6-fold symmetry, and in it each subunit is composed of a tetra-

hedral arrangement of four units (each with a mass of 54,000 daltons and two heme groups) arranged so that a a-fold axis of the tetrahedron is perpendicular to the plane of the ring. The rings are then stacked head to head (or tail to tail) so that the apices of the tetrahedra in each ring are in contact at the equa- torial plane of the molecule. This arrangement would be compatible with the observation that, in the intact molecule, the subunit has a 3-fold axis of symmetry, and it would also explain the apparent protein bridge between the two tiers of rings described by Levin (37).

In any event, I conclude that the hemoglobin molecule of A. cristata has 96 heme groups and 192 polypeptide chains.

It should be pointed out here that several other hemoglobins resemble that of Arenicola. Both Shlom and Vinogradov (42) and Rossi-Fanelli et al. (41) have found similar ratios of heme to protein for Lumbricus hemoglobin as have Chew et al. (43) for the hemoglobin of Marphysa sanguinea. In addition for the chloro- cruorin from Spirographis spallanzanii, Antonini, Rossi-Fanelli, and Caputo (44) reported a value of 1 mole of chloroheme per 35,000 g of protein. If in addition to the similarity in heme content, there is also some similarity between the protein structure of these hemoglobins and that of A. cristata, then one must surmise that the notion of one heme per polypeptide chain, as expressed by Svedberg (36), does not apply to this class of proteins. In any event the value of one heme per 17,250 g of protein for A. marina (45) is probably incorrect.

The most intriguing aspect of such molecules, of course, is the mechanism of cooperativity. Even though the value of n in the Hill equation is large (n = 6), the interaction energy per heme group is only 1800 cal per mole (as compared to approximately 3000 cal per mole for human hemoglobin). This means that the cooperative unit may be the whole molecule, or only a part of it, such as one ring or one 10 S subunit. In the latter case, the system would resemble human hemoglobin in that the Hill coefficient would be 6 for the 8-heme unit, as opposed to 3 for the tetrameric molecule. Weber (46), in fact, has reported that for A. marina, cooperativity is not transferred across the equa- torial plane, and must therefore reside primarily in the 10 S subunit. His conclusions, however, are open to alternate inter- pretation because of the difficulties inherent in obtaining and performing oxygenation experiments on the breakdown products. The function of the various subunits of the molecule, including the 55,000 dalton unit with two heme groups, remains to be fully described. It is clear that knowledge about this intriguing question would do much to shape current thinking not only about the nature of cooperativity, but also how molecular architecture may be correlated with function.

Acknowledgments-I would like to thank Guido Guidotti for his advice and encouragement. I am also grateful to Elizabeth Mains for the electron micrograph shown in this paper.

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Lloyd WaxmanArenicola cristataThe Hemoglobin of

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