On the structure of sperm whale myoglobin

J. Mol. Biol. (1962) 5, 663-682

On the Structure of Sperm Whale Myoglobin I. The Amino Acid Composition and Terminal Groups of the Chromatographically Purified Protein A. B.

EDMUNDSON

The Rockefeller Institute, New York 21, New York, U.S.A. and the Laboratory of Molecular Biology, Hills Road, Cambridge, England AND

C.R. W.Rms Department of Biology, Brookhaven National Laboratory, Upton, Long Island New York, U.S.A . (R eceived 28 August 1962) The hemoprotein t hat comprises 96 % by weight of the protein in cryst alline sperm whale m y oglobin may be re solved into at least five components b y chromatography on t he carboxylic resin IRQ-50. Four of the five heme-containing components (representing 92 % of the prot ein) po ssess identical amino acid compositions; t he fifth compone n t has not been obtained fr ee of non-heme protein but appe ars t o be very sim ila r to, if not id en ti cal wi th, t he com posit ion of the other h eme-con taining comp one nts. The amino ac id analyses have shown tha t the m yoglobin molecul e contains 153 amino a cid residues and possesses a mol ecular weight of 17,816. Sixty-five per cent of the hem e-containing protein is represented by two of t he com po ne n ts, designated as components IV and V, and these have been isolat ed in quantity for st r uctural study . Quantitative methods have be en us ed t o d em ons trat e that components IV and V of sp erm whale myoglobin contain a single polypeptide chain wit h valine at the aminoterminus and glutamine at the carboxy l-t erm inus.

1. Introduction Myoglobin crystallizes from concentrated solutions of ammonium sulfate or sodium and potassium phosphate buffers, a property which has led to an extensive X-ray crystallographic st udy of the structure of sperm whale myoglobin by Kendrew et al. (1958, 1960, 1961). I somorphous heavy at om derivatives of sperm whale myoglobin can be prepared with several simple reagents (Kendrew, Parrish, Marrack & Orlans, 1954). A sufficient number of X -ray reflections from crystals of these derivatives were measured by the Cambridge group to permit the calculation of a three-dimensional Fourier synthesis of the electron density in the unit cell, initially to a resolution of 6 A (Kendrew et al., 1958) and more recently to a resolution of 2 A (Kendrew et al., 1960). The results obtained in the latter synthesis are still being analysed in detail, though the outlines of the secondary structure are perfectly clear and a tentative amino acid sequence has been deduced (Kendrew et al., 1961). 663

664

A. B. EDMUNDSON AND C. H. W. HIRS

At an early stage in the X-ray attack the desirability of simultaneous chemical studies on the primary structure of the protein became apparent and these were initiated at the suggestion of Dr. Kendrew. The present communication and those that follow describe the preliminary phases of the chemical approach, which is being continued by one of us at Cambridge. The results obtained thus far are of significance because they have been of considerable value in the interpretation of the electron density projections and because, in a wider sense, they are pertinent to studies on the structure of hemoglobin (Perutz et al., 1960; Braunitzer et al., 1961; Konigsberg, Guidotti & Hill, 1961) in particular, and of other globular proteins in general. The first objective in the chemical studies was the establishment of the amino acid composition of sperm whale myoglobin. The results of this work form the subject of this report. Before detailed amino acid analyses were undertaken, attention was directed to the known inhomogeneity of crystalline sperm whale myoglobin. Bodo & Kendrew (personal communication) resolved the crystalline protein into at least four hemecontaining components by chromatography on the sodium form of IRC-50. The nature of the structural variations responsible for the difference in chromatographic properties of these components subsequently became of interest when the secondary and tertiary structures of the protein were defined by X-ray diffraction measurements on crystalline myoglobin that undoubtedly represented a mixture of these components. In the present studies crystalline sperm whale myoglobin was chromatographed over the sodium form of IRC-50 under conditions similar to those used by Bodo & Kendrew. EVidence for the existence of at least five heme-containing components was found and four of these components were isolated in purified form. The major chromatographic fraction, composed of a mixture of two proteins designated as myoglobin IV and myoglobin V, represented approximately 65% of the total protein in crystalline sperm whale myoglobin and was studied in detail. It subsequently served as the starting material for the degradative work. Preliminary experiments by Ingram (1956) with crystalline sperm whale myoglobin suggested that the protein contains a single polypeptide chain and that valine is the amino-terminal residue of the chain. A more detailed study of this question was undertaken in the present work which has included experiments aimed at revealing the amino acids at both the amino- and carboxyl-termini. The results have substantiated Ingram's conclusion and have, in the meantime, received independent verification from the X-ray structural work at a resolution of 2 A (Kendrew et al., 1960; Kendrew et al., 1961).

2. Experimental (a) Crystalline sperm whale myoglobin

To ensure correspondence of results, Dr. J. C. Kendrew kindly furnished all the crystalline myoglobin used in this work. The preparations supplied consisted of an ammonium sulfate-myoglobin filter paste, containing 40 to 45% myoglobin, isolated from the skeletal muscle of sperm whale by the procedure of Kendrew & Parrish (1956). (b) Preparation of I RC-50 for myoglobin fractionation

The sodium form of the resin in particles ranging in "diameter" from 25 to 50 II- is required for effective chromatography of the protein. Such material may be prepared from both Amberlite XE-64 (IRC-50) "micropowder," (Rohm and Haas Co., Philadelphia, Pa.), and from Amberlite IRC-50 Type III, manufactured by the Rohm and Haas Co. and

COMPOSITION OF SPERM WHALE MYOGLOBIN

665

available through the Fischer Scientific Co., New York, N.Y. Fractionation of the resin may be achieved by the flotation method of Hamilton (1958). In the apparatus currently used, the sodium form of the resin is introduced in portions of 200 ml. into a conical 2 liter separatory funnel serving as the flotation chamber. Initially, tap water is passed through the chamber at a rate of 50 ml.jmin to remove particles with "diameters" less than about 25fL. Thereafter an increase in the flow rate to 120 ml.jmin removes a fraction consisting predominantly of particles with "diameters" in the desired range. The yield of resin suitable for the chromatography of myoglobin has never been less than about 10% and is usually approximately 30 %. Following flotation, the resin is freed of polyvalent cations adsorbed from the processing water by exhaustive washing with 4 N-HCI. Excess acid is washed away with water and the resin is converted to the sodium form with 2 N·NaOH, washed with water and stored in the cold. To obtain columns which continue to flow at a constant rate it has been of particular importance to remove small particles ("fines") of IRC·50 by decantation with water. (c) Eluents for the chromatography of myoglobin Experience has demonstrated that the elution of myoglobin from IRC-50 is sensitive even to slight variations in the composition of the sodium citrate buffers used for chromatography. In practice, the solutions listed in Table 1 are made up in bulk, and a test chromatogram is obtained with each new lot of buffer. On the basis of the test chromatogram final adjustment of the buffer composition is made in order to duplicate the effluent patterns shown in Fig. 1: the adjustments are made by adding citric acid or sodium hydroxide in portions of 2 g until the desired chromatographic performance is obtained. Following this adjustment, the effluent patterns shown in Fig. 1 will be obtained repeatedly from each new lot of buffer. It is for these reasons that the pH values quoted in Table I should be regarded as significant only in terms of the differences between solutions. TABLE

1

Composition of sodium citrate-chloride buffers most frequently employed for the chromatography of myoglobin x-Sodium

pH (at 25°C)

Citric acid

hydroxide (carbonate free)

Sodium chloride

Volume

5·86 5·88

210·0 g 210·0

2712 ml, 2722

40·2 g 39·6

101. 10

(d) Chromatography of myoglobin on lRO-50 (1) Columns were prepared by the procedure of Hirs (1955). The following representative description applies to the use of a column 4 x 15 cm. One g of myoglobin-ammonium sulfate filter paste is dissolved in 5·0 ml. of water (at 4°0) and the pH is maintained near neutrality by the addition of 0·10 ml. of 4 N.K 2HP04 • Solid sodium dithionite is added gradually until examination of the spectrum in a Hartridge reversion spectroscope reveals that all the myoglobin has been reduced. The carboxy derivative is formed by passing carbon monoxide through the solution for 3 min. The resulting solution of carboxymyoglobin is dialysed at between 2 and 4°C against 4 portions of tho buffer used for chromatography in a thin-film apparatus similar to that described by King & Craig (1955). The buffer is kept saturated and stirred with a slow stream of carbon monoxide. (2) The dialysed solution is allowed to enter the column without application of external pressure. Elution is allowed to proceed at 5 em/hr. The effluent is collected in portions of 6 ml, in calibrated spectrophotometer tubes. The progress of the fractionation may be followed visually, as well as by direct measurements of the absorbancy of the effluent fractions at 579 mfL with a Coleman junior spectrophotometer. The carboxymyoglobin is dissociated by light. As a result, the absorbancy of the effluent containing the protein diminishes with time and it is therefore not possible to determine recoveries of the protein by measurements at 579 mfL'

666

A. B. EDMUNDSON AND C. H. W. HIRS (e) Isolation of protein fractions

The combined effluent corresponding to each chromatographic fraction is successively dialysed at 4°C against 100 vol. water containing 2 ml. concentrated NH 40Hjl., against a similar volume of water containing 0·1 ml. concentrated NH 40Hjl., and, finally, against water. The protein solution is lyophilized. The resulting powders are subjected to analyses for ash and moisture content. If, on the basis of such analyses, the product is found to contain more than 5% ash, the dialysis is repeated. A second series of dialysis and lyophilization steps, however, is invariably associated with a decrease in the solubility of the protein fractions in water. (f) Preparation of globin from myoglobin

The procedure used was patterned after that of Theorell and Akeson (1955). A 1% solution of myoglobin in water at 4°C was added to 20 vol. acetone-HCl (2 ml. N.HCIJl. of acetone) at 4°C. The precipitate was collected by centrifugation in the cold and then dissolved in water to give a 4% solution. After filtration to remove a small quantity of denatured myoglobin the solution was dialysed successively against 0·1 N-NaHCO s and water. The globin was obtained as a lyophilized powder. (g) Amino acid analyses

(1) Acid hydrolysates of the proteins have been prepared at 110°C in evacuated, sealed tubes from approximately 4 mg of sample in 0·5 ml. of twice-distilled constant boiling HCl of known ammonia content. The amino acid composition of the hydrolysates was determined by the method of Moore, Spackman & Stein (1958) with the aid of an instrument of the type described by Spackman, Stein & Moore (1958). The procedure described by Hirs, Stein & Moore (1954) was used to bring the amino acid hydrochloride residue from the evaporated hydrolysates into solution. After the first analyses of the protein had demonstrated that sperm whale myoglobin does not contain cystine or cysteine, the oxidation step in the procedure was omitted. (2) Tryptophan was determined by the starch column method of Moore & Stein (1949) on alkaline hydrolysates prepared by the starch-barium hydroxide procedure described by Dreze (1956). (h) Reaction with I-fluoro-2,4-dinitrobenzene

The pH-stat method of Levy (1954) was used. Approximately 10/Lmoles of protein were suspended in 10 ml. of water at 40°C, and the protein was brought into solution by increasing the pH to 9·0 with standard 0·1 N-NaOH. The reaction was initiated by addition of 0·2 ml. (1·2 millimoles) of fluorodinitrobenzene (Eastman) and stirring was continued until the rate of uptake of alkali was constant (cf. Fig. 3). The reaction was accompanied by precipitation of the protein. Excess reagent and dinitrophenol formed from it by hydrolysis were removed by continuous extraction with ether for 8 hr at about pH 5. The protein was collected by centrifugation. Dissolved ether and salts were removed by dialysis. (i) Determination of dinitrophenyl end-groups

(1) Samples of the protein derivative (15 to 20 mg) were subjected to hydrolysis in constant boiling HCI at 110°C in evacuated, sealed tubes for 6, 12, 18 and 24 hr. Excess acid was removed by repeated concentration under reduced pressure on a rotary evaporator (bath temperature 35°C). The residue was in each instance dissolved in 30 ml. of water and the solution was extracted continuously with ether for 18 hr. Analyses were performed on portions of the aqueous fraction and the ether extract as described below. (2) The aqueous fraction was evaporated to dryness. When the hydrolysis time was less than 18 hr, the residue was dissolved in constant boiling HCI and the solution was heated at 110°C in an evacuated, sealed tube until the total heating time for that sample had reached 24 or 48 hr. Thereupon the excess acid was removed and the residue was subjected to quantitative amino acid analysis as described in (g) (1).

COMPOSITION OF SPERM WHALE MYOGLOBIN

667

(3) The ethereal solution was dried with anhydrous sodium sulfate and was concentrated to dryness. Excess dinitrophenol was removed by sublimation as described by Mills (1952). Two procedures were used to identify DNP-amino acids, paper chromatography and hydrolysis by the method of Mills (1952) to form free amino acids, which were determined with the aid of an amino acid analyser. For paper chromatography the ascending procedure of Blackburn & Lowther (1951) was used, particularly the solvent system based on tert:» amyl alcohol and phthalate buffer at pH 5. For conversion of the dinitrophenylamino acids into free amino acids, a portion of the acetone solution was dried in an ignition tube, 0·5 ml. of saturated baryta was added, and the tube was evacuated and sealed. After heating at 100°C for 1·5 hr, the hydrolysate was cooled and the barium ion precipitated as carbonate by saturating the solution with carbon dioxide. The barium carbonate was separated by centrifugation and washing, and the aqueous portion was concentrated to dryness. The residue was subjected to amino acid analysis. (j) Degradation of myoglobin with carboxypeptidase (1) The enzyme was a twice-crystallized preparation treated with di-isopropylphosphorofluoridate and was obtained from a commercial source (Worthington Biochemical Corp., Freehold, N. J. lot number CO 575 DFP). The enzyme preparation undoubtedly contained both carboxypeptidase A and B. Solutions of the enzyme in 2 M-NaCI were prepared as described elsewhere (cf. Hirs, Moore & Stein, 1960). (2) Between 10 and 20 mg of myoglobin were dissolved in 0·5 to 1·0 ml. of 0·1 M-tris-HCI buffer at pH 8·3. The solution was maintained at 40°C. In a typical experiment the hydrolysis was initiated by addition of 0·1 ml. of a solution containing 720 fLg enzyme nitrogen/ml. (final enzyme concentration, 162 fLg enzyme nitrogen/ml.). The reaction was terminated by addition of 0·5 ml. of 15% trichloroacetic acid. After removal of the precipitate and extraction of trichloroacetic acid, portions of the solution were subjected to amino acid analysis. The analyses were conducted at 50°C throughout, under which conditions the amides, asparagine and glutamine, emerge as a single band at the serine position on the columns used for the determination of the acidic and neutral amino acids. A separate determination of serine would have required the performance of parallel runs with columns operated at 30°C. Such determinations were not carried out because, as will become evident in an accompanying paper (Edmundson & Hirs, 1962a), serine is not part of the carboxyl-terminal sequence in sperm whale myoglobin. The experiments with the intact protein were performed to demonstrate that the same pattern of release of free amino acids is observed with myoglobin as with the carboxyl-terminal peptide from the trypsin series of peptides.

3. Results (a) Ohromatography of sperm whale myoglobin Effiuent curves obtained on chromatography of the crystalline protein in the form of its carboxy (CO) derivative at various pH values in the range 5·82 to 5·92 are presented in Fig. 1. The curves demonstrate the sensitivity of the separations to small changes in the composition of the eluent. Thus, at pH 5·92 the components move relatively rapidly, with poor resolution; at pH 5·82 they move much more slowly, with partial resolution; and at pH values lower than 5·82 they are strongly adsorbed on the resin. The heme-containing components of crystalline myoglobin were recovered in a total yield of approximately 95% (by gravimetric determination) in the pH range between pH 5·82 and 5·92; at pH 5·82 chromatography on IRC-50 was accomplished with a recovery of heme-containing material closer to 90%. The colorless (non-heme) component(s) of crystalline myoglobin migrated on the column faster than any of the heme- containing components. They could be detected in the effiuent with the aid of ninhydrin (Hirs, 1955) or by measurement ofthe absorbancy ofthe effiuent at 280 txu». For clarity only the measurements obtained at 579 mp. are shown in Fig. 1. 43

A. B . EDMUNDSON AN D C. H . W. HIRS

668

At pH 5·88 carboxymyoglobin was resolved into at least five heme-containing components. Component V appeared as a distinct red band on the column but, because of tailing, was observed only as a shoulder on peak IV in the effluent curve. The same band was also observed on ot her columns of t he same dimensions, 4 x 15 em, operated at pH 5,86, but was not usually detected as a definite shoulder in the spectrophotometric measure ments.

pH 5·92 c-,

v

c:

ill

0 .D

0on .D «

0'6

0

0

4

pH 5·86

5

pH 5·82

>v c: 0

L..

0on

IV

N

.D

0'6

.D

-c 0

300

ml.

ml.

FIG. 1. Chromatography of carboxymyoglobin on IRC -50. The resin was in the sodium form, equilibrated with 0·34 N-sodium citrate-chloride b uffer in the pH range from 5·82 to 5·92 (as measured at 25 °0) . The eluents were saturated with carbon monoxide and the chromatograms were obtained at 4°C. Column dimensions in all experiments, 4 x 15 em. The e£lluent patterns sh own were obtained by measurement of the a bsor b ancy of the e£lluent fractions (vo lume 6·0 ml.) in calibrated photometer tubes with a light path of 18 mID, at 579 ttu», the absorption maximum of the ",·band of carboxymyoglobin. Flow ra t e 5 em /hr. The discontinuity of curves I , 3 and 5 is explained in the text. The designation of the peaks I and II of curve 1 is arbitrary, as it is possible that peaks I and II of curve I correspond to peaks I , II and III in the remaining chromatograms.

The chromatographic homogeneit y of t he five components separated in an experiment of the kind represent ed by curve 3 in Fig. 1 was proved by removing appropriat e cuts containing these components from the effluent fractions . In each case the protein was isola ted by dialysis and lyophilization, after whieh a portion of the powder obt ain ed from each cut was subjected to re chromatography on the column t hat had been used for the original separation. The results are shown in Fig. 2, and demonst rat e that, when allowance is made for tailing, each of the components of the crystalline protein behave as individual molecular species. Components IV and V as a composite fraction were isolated after chromatography at pH 5·88. The fractions corresponding to the first 10% of the peak designated as IV + V in Fig. 1(3) were discarded in order to minimize contamination by component III; usually, the fractions corresponding to the last 15% of this peak were also discarded. The yield of lyophilized fraction I V + V recovered has generally been 250 mg per g of crystalline filter paste. If the contribution of the discarded fractions is

COMPOSITION OF SPERM WHALE MYOGLOBIN

669

allowed for, components IV + V represent 65 to 70% of the heme-containing proteins in the effluent, while the minor components, Pre-I, I, II and III account for I, 2 to 3, 7 to 10 and 20%, respectively.

c-,

v

2 0'0

t

...... _ _...... ~-""----""----'-----

c o

10·:lb3~

_ _"""

""':'_~_-----l_~~-.! ::a;:

300

600

:=z:s:J 900

1200

ml.

FIG. 2. Chromatography of carboxymyoglobin at 4°C on a column of the sodium form of IRe·50, 4 x 30 ern, with 0·34 N-sodium citrate-chloride buffer saturated with carbon monoxide as eluent. The pH, as measured at 25°C, was 5·88. The results of rechromatography of the protein corresponding to peaks I, II, III and IV + V is indicated by curves 1 through 4. cf. Fig. I for other details.

Bands of the minor components were apparently well separated in the pH range from 5·82 to 5·90. However, the effluent curves do not return to the baseline between adjacent peaks because of the tailing that characterizes the chromatography of these proteins on IRC-50. The discontinuity in the effiuent curve just below Roman numeral V in Fig. 1(3) (cf. also Fig. 1(1) and 1(5)) was occasioned by the circumstance that an interval of 12 hours separated readings between these two points. The decrease in absorbancy in this period is attributable to the photolytic dissociation of the carboxy derivative of the protein. Difficulty was experienced when attempts were made to prepare useful quantities of the minor components in uncontaminated form by chromatography on columns 4 x 15 em at pH 5·88. To obviate this problem the more recent work has been performed with columns 7·8 x 15 em at pH 5,86, which are capable of dealing with a 5 g charge of crystalline filter paste, and with which more complete resolution of component V is achieved. (b) Amino acid composition of components I V + V

A number of different preparations of components IV + V and of the globin derived from them by acetone-HCI cleavage were subjected to quantitative amino acid analysis. In the Tables that follow the analytical values obtained have all been corrected for the moisture and ash content of the preparation analysed. The ash derived from the iron of the protein (assumed to appear as ferric oxide when present) has not been included in the ash correction. The results, expressed as grams of amino acid per 100 g of anhydrous, ash-free protein, obtained on analysis of a single sample of components IV + V, are presented in

A. B. EDMUNDSON AND C. H. W. HIES

670

TABLE

2

Amino acid analyses of hydrolysates of myoglobin components IV + V Grams amino acid per 100 g protein Amino acid Time of hydrolysis (hr) 22 22 72 72 Aspartic acid 5·52 5·57 5'54 5·36 Glutamic acid 13·7 14·3 14·3 14·1 Glycine 4·20 4·40 4·21 4·35 Alanine 7·76 7·80 8·03 7·84 Valine (4'72) (4-81) 5'02 5·02 Leucine 12·0 12·3 12·3 12·0 Isoleucine 5,99" (5'00) (5'69) 6'04a Serine 2·73 2·84 2·04 2·H Threonine 2·94 3'01 2·61 2·56 Methionine 1·47° 1·51° 1·53° 1·47° 2·38 Proline (2'67) 2·36 2·17 Phenylalanine 5·01 5·29 4·99 4·93 Tyrosine 2'52 d 2·66d 2'73d 2·46d Histidine 9·18 9·44 9·28 9·07 14·1 Lysine 14·3 14·1 14·1 Arginine 3·53 3·65 3·62 3·50 Tryptophan Amide ammonia 0·76 0·72 0·92 0·85 Sub-total Heme Total

Amino acid residues Average per 100 g protein g 5·50 14·1 4·29 7·86 5·02 12·2 6,02" 3·18 b 3·17 b 1·50° 2·30 5·06 2'59 d 9·24 14·2 3·58 1.95 6 0·63 f 102·4 3·14 105·5

Calculated Number of Nas % number of residues for residues of mol. wt. to nearest total N integer of 18,000

4·76 12·4 3·26 6·27 4·25 10·5 5·20 2·63 2·69 1·32 1·94 4·51 2·33 8·17 12·5 3·21 1·78

3·65 8·47 5'05 7·80 3·79 8·22 4·06 2·67 2·35 0·89 1·77 2·71 1·26 15·8 17·2 7·26 1·67 3·3

87·7 3·14 90·9

97·9 1·80 99·7

8'04 18·7 H·l 17·2 8·36 18·1 8·94 5·90 5·18 1·97 3·90 5·96 2·79 H·6 18·9 4·01 1-86 7·2

8 19 H 17 8 18 9 6 5 2 4 6 3 12 19 4 2 (7)C 153h

Values are calculated for the anhydrous, ash-free protein. The averages do not include the values in parentheses. a Isoleucine values are corrected for formation of 1 to 2% alloisoleucine, b Extrapolated values for serine and threonine, which decompose during hydrolysis. c Methionine values corrected for the formation of methionine sulfoxides. d Tyrosine values corrected for the formation of chlorotyrosines. e The tryptophan value represents the average of two independent determinations. f The value for amide ammonia is corrected for the quantity of ammonia formed in the decomposition of serine and threonine. There is no correction for the negligible quantity of ammonia in the reagents. Il Not included in totals. h On the basis of the integral number of residues and with one heme per molecule the molecular weight calculated is 17,816. This includes the amide groups and the assumption that the protein contains a single polypeptide chain.

Table 2. With the exception of the values for tryptophan and those amino acids undergoing extensive destruction or incomplete liberation on acid hydrolysis, the values in Table 2 show deviations less than ± 3% from the average values. The procedures preceding the chromatographic analysis were therefore kept constant to the extent that the reproducibility was essentially within the experimental error (± 3%) associated with the analytical method alone. From the average corrected recoveries in Table 2 the molar ratios of the stable amino acids, arginine, phenylalanine, aspartic acid, glycine, alanine, leucine, glutamic acid and lysine were calculated and found to approximate closely the following integral values, respectively: 4 : 6 : 8 : 11 : 17 : 18 : 19 : 19. These values and the corresponding corrected recoveries

TABLE 3 Amino acid analyses of hydrolysates of the globin from myoglobin components 1 V + V

a Gr am s amino aci d pe r 100 g prot ein Average

Amino a cid 22

22

5·8 4 15·6 4·48 8·36 4· 99 12·9 (5' 95) 2·90 3·06 1·64 c 2·71 5·46 3·07 d 10·4 15·9 3·85 0·82

5·67 15·4 4·41 8· 19 4·98 12·6 (5'88) 2·82 3·00 1·64 c 2 ·53 5·48 2·95d 9·90 15·1 3·69 0· 83

Time of hydrolysis (hr) 24 72 72

72

72

5·83 15· 5 4·60 8·26 5·0 1 12·7 6·35 " (2-41) 2·69 1·54° 2·6 1 5·4 1 2·98 d 10·1 15· 6 3·70 1·09

5' 76 15'7 4·46 8'29 5·06 12·8 6·46" 1·97 2·62 1·50 c 2·66 5'32 3·04 d 10·2 15·8 3·84 1·05

Amino ac id residues per 100 g protein g

N as % of t otal N

Number Calcula ted of n umber of re sid ues residues to nearest in teger

0

is:

t'd 0

rn H

1-3 H

Aspartic acid Glutam ic a cid Glycine Alanine Valine L eu cine I soleu ci ne Se rine T h reoni ne Methioni ne Proline P he nylalanine Tyrosine H ist idi ne Lysine Argini ne Amide ammonia Total(l)

5·76 15·7 4·47 8·28 5· 04 13·0 (6'14) 2·742·93 1·69c 2·70 5·46 2·94d 10·1 15·7 3·73 0· 82

5·71 15·4 4' 42 8· 16 4·98 12·8 6·35" 2·00 2·62 1·59c 2'5 4 5·34 2' 77d 10·1 15·3 3·63 1·34-

5·66 15·5 4·42 8· 17 5·04 13·0 6·28" 2·02 2·59 1·56c 2·55 5·30 2·9 1d 10·1 15·6 3·82

H O

5 ·75 15·5 4·47 8·24 5·01 12·8 6·36" 3·35b 3·22b 1·59c 2·6 1 5·40 2·95 d 10·1 15·6 3·75 0·72f 107·4

4·97 13·6 3·40 6·58 4·24

ll·O 5·49 2·77 2·73 1·40 2·20 4·80 2·6 5 8·93 13·7 3·36 91·8

3' 57 8·72 4·93 7·65 3'5 4 8·08 4·01 2·64 2·24 0·88 1·88 2· 70 1·35 16·2 17·7 7·12 3·5 96 ·7

7·97 19·4

ll ·O 17·1 7·88 18·0 8·95 5·89 4·98 1·97 4·19 6·02 3·01 12·0 19·7 3·97 7·8

8 19 II 17 8 18 9 6 5 2 4 6 3 12 20 4(8)11' 152

0

Z

0 ":l rn t'd t.".J ~

is: ~ ~ ~

t'" t.".J

is: ~

0

o

t'"

0

td

H

Values are calcula ted for the anhydrous, ash-free pr ot ein. The a verages do not include the values in parentheses . (I) The totals do not inolude t he contrib u t ion fr om the two tryptophan residues known to be p resen t (cf. Table 2) . Other footnotes a re the same as those used in reference to the contents of Table 2.

Z

'" -> ....

672

A. B. EDMUNDSON AND C. H. W. HIRS

were used to calculate a minimum molar equivalent for each amino acid. The average minimum molar equivalent derived from these values was subsequently used to calculate the molar ratios for all the amino acids shown in Table 2. Analyses were also performed with a sample of globin prepared from components IV + V. The results obtained after independent hydrolyses for periods of 22 (or 24) and 72 hours are presented in Table 3. In general the reproducibility attained in these analyses was better than that attained in analyses of the parent proteins. Table 2 indicates that myoglobins IV + V contain 153 amino acid residues and 7 amide groups. The molecular weight based on this composition, together with the contributions of the heme group and the terminal molecule of water, is 17,816. The nitrogen recovery in this series of analyses of components IV + V was 99'7 % if the contribution of one heme group is included in the calculation. The weight recovery, however, was 90·9% (with the heme group included). The low weight recovery is probably attributable to the binding of anions by the proteins during recovery from the column eluate. If it is assumed that citrate ions are bound by the proteins, a weight recovery of 91% on analyses for amino acids would be accounted for by the binding of 9 citrate ions per molecule. Low weight recoveries have similarly been observed in the amino acid analysis of calf thymus histones (Crampton, Moore & Stein, 1955) and the corticotropins (Levy, Geschwind & Li, 1955). Comparison with the amino acid composition of components IV + V (Table 2) with the composition of the globin derived from them (Table 3) reveals excellent agreement for the stable amino acids . In assigning integral values to the calculated totals of each residue the lysine values in myoglobin approximate 19 residues, while in the globin analyses the values more closely approximate 20 residues. In view of the relatively smaller deviation from the mean observed in the analyses for lysine in myoglobin, a value of 19 is more probably correct; but a clear-cut decision would require a greater precision in measurement than the methods used are presently capable of attaining. Analysis of 120-hour hydrolysates of two preparations of myoglobins IV + V showed that the liberation of valine and isoleucine is complete in 72 hours. Table 3 demonstrates that on analysis of globin an essentially quantitative recovery of the nitrogen was attained when the two tryptophan residues known to be present, but not determined in globin, are included in the calculation. However, as with myoglobins IV + V, the weight recovery was low and could be accounted for by the binding of 6 citrate ions per molecule of protein. (c) Amino acid composition of different preparations of myoglobins IV + V

To demonstrate the reproducibility of the chromatographic procedure used for the separation of components IV + V from crystalline myoglobin, several preparations of components IV + V derived from different lots of crystalline sperm whale myoglobin were subjected to quantitative amino acid analysis. The results of such analyses showed that different preparations of components IV + V are indistinguishable in terms of over-all amino acid composition. (d) Amino acid composition of minor components of crystalline sperm whale myoglobin

A small quantity of the colorless protein moving more rapidly on the IRC-50 columns than component I was obtained. On amino acid analysis this colorless material

COMPOSITION OF SPERM WHALE MYOGLOBIN

673

proved to possess a composition markedly different from that of the heme-containing components and it was therefore not studied further. The results obtained upon analysis of a preparation of component I are presented in Table 4. The amino acid compositions of components II, III, and V are similarly TABLE

4

Amino acid analyses of hydrolysates of myoglobin component 1 Grams amino acid per 100 g protein Amino acid Time of hydrolysis (hr) 22 22 72 76 Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Methionine Proline Phenylalanine Tyrosine Histidine Lysine Arginine Tryptophan Total1

3·24 6·92 2·21 3·55 2·42 5·61 (2'52) 1·59 1·64 0·743° 1·59 2·59 1·66 d 4·19 6·56 1·95

3·29 3'29 7·14 6·88 2·26 2'20 3·67 3'57 2·44 (2'33) 5·83 5·72 (2'74) 2'81 a 1·56 1'02 1·57 1'35 (0'868) 0·750° 1·68 1-59 2·69 2·57 l·Ud l'39 d 4·21 4·35 6·74 6·48 1·91 2·00

3·22 6·97 2·22 3·57 2·45 5·68 2·79 a 1·01 1·33 0·768° 1·56 2·60 1·56d 4·16 6·52 1·92

Amino acid Number residues Calculated of Average per 100 g number of residues protein residues to nearest integer g 3·26 6·98 2·22 3·59 2·44 5·71

a-so1·92 b 1·75 b 0·754° 1·61 2·61 l'81 d,l 4·23 6·58 1·95 0·727 k 50·9

2·82 6·13 1·69 2·86 2·06 4·93 2·42 1·59 1·49 0·664 1·36 2·33 1·63 3·74 5·78 1·75 0·664 43·9

9·08 17·6 1l·0 14·9 7·71 15·8 7·89 6·78 5·45 1·87 5·19 5·85 3·70 10·1 16·7 4·15 1·32

9 18 II

15 8 16 8 7 5 2 5 6 4 10 17 4 1 146

Values are calculated for the anhydrous, ash-free protein. The averages do not include the values in parentheses. l Extrapolated value. k The tryptophan value represents one independent determination. 1 The totals do not include the values for the heme group. The low recoveries were occasioned by incomplete removal of salts at the dialysis step, and the difference in equivalent weights of the salts present before and after ashing. (For footnotes (a) to (d) see Table 2.)

derived in Tables 5, 6 and 7, in which the molar ratios for the constituent amino acids were calculated in the manner described for components IV + V. The tryptophan values presented in the Tables are based on single independent determinations (cf. (h) (2)). The composition found for component V permits the deduction of the composition of component IV. The complete results show that with the exception of component I the amino acid compositions of the remaining components of crystalline myoglobin are all identical. The composition derived for component I is similar to that ofthe other components. In view of the incomplete separation of component I from the colorless protein(s) moving more rapidly on the IRe-50 columns it is probable that the difference in composition of component I is due to contamination, and that component I in reality has the same composition as the other constituents.

674

A . B. EDMUNDSON AND C . H. W. HIRS TABLE 5 Amino acid analyses of hydrolysates of myoglobin component II

Grams amino acid per 100 g protein

Amino acid

Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Methionine Proline Phenylalanine Tyrosine Histidine Lysine Arginine Tryptophan

Number Amino acid residues Calculated of Average per 100 g number of residues to nesrest Time of hydrolysis protein residues 24 hr integer 70 hr g 5·01 12· 7 3·81 6·84 4·28 10·6 (5'06) 2·64 2·64 1·42 C 2·27 4·60 2·49 d 8·46 12· 7 3·32

5·01 12' 7 3·88 6·87 4·29 10·9 5 '56" 2'09 2·40 1·25 c 2·03 4'73 2·40 d 8'56 1301 3'34

Total'

5·01 12·7 3·85 6'86 4·29 10·8 5·56" 2·97 b 2'77b 1·34 c 2·15 4·67 2·45d 8·51 12·9 3·33 1·62k

4·33 11·2 2·93 5·48 3·63 9·32 4·80 2·46 2·35 1-18 1·81 4·16 2·21 7·52 11·3 2·98 1· 48

91·8

79·2

8 19 11 17 8 18 9 6 5 2 4 6 3 12 19 4 2

8·18 18·8 11·2 16·7 7·96 17·9 9·22 6·13 4·87 1·95 4·06 6· 15 2·94 11·9 19·2 4·15 1·72

153

Values are calc ulated for the anhydrous, as h-free protein. The averages do not include the value in parentheses. k The tryptophan value represents one independent determination. J The totals do not include values for the heme gro up. The low recoveries were occasioned by incomplete removal of salts at the dialysis step, and by the difference in equivalent weights of (For f ootnotes (a) to (d) see Table 2). the salts present before and after ashing. TABLE 6 Amino acid analyses of hydrolysates of myoglobin component III

Grams amino acid per 100 g pro t ein Amino acid Time of hydrolysis (hr) 24 22 72 72 Aspartic acid Glutamic acid Glycine Alanine Valine L eucine I soleucine Serine Threonine Methionine Proline P henylalan ine Tyrosine Histidine Lysine Arginine Tryptophan Total'

4·84 4·80 4·87 4·84 12·9 13·0 12·7 12·9 3·77 3·69 3'76 3·75 6·93 6·75 6·91 6·86 4·21 (3'98 ) 4·10 4· 11 10·6 10·5 10' 7 10·7 (4'77) (4'93) 5· 30" 5 ·32" 2·40 1·64 2·47 1·62 2·56 2·56 2·18 2· 13 1·28 c 1·29 c 1·26 c 1·29 c 2·23 2·15 2·11 2·23 4·47 4·48 4·45 4·47 2' 38d 2·47 d 2·36 d (2'20) 8·18 8·20 7·97 8·24 12·5 12·2 12·6 12·4 3· 13 3·03 3·13 3·05

Number Amino acid Calculated of residues Average per 100 g number of residues protein residues to nearest integer g 4·84 12·9 3·74 6·86 4· 14 10· 6 5,31" 2 '97b 2·76 b 1·28c 2·18 4·47 2·40 d 8·15 12·4 3·09 1·47 k

4·19 11·3 2·84 5·48 3·50 9·15 4·58 2·46 2·34 1·13 1·84 3·98 2·16 7·20 10·9 2·77 1·34

89·6

77-1

8·07 19·4 11·0 17· 1 7·83 17·9 8· 98 6·27 5·15 1·90 4·19 5·98 2·94 11·6 18·8 3·93 1·60

8 19 11 17 8 18 9 6 5 2 4 6 3 12 19 4 2 153

Values are calc ulated for the anhydrous, ash-free protein. The averages do not include the values in parentheses. k The tryptophan value represents one independent determination. J The totals do not include values for the heme group. The low recoveries were occasioned by incomplete removal of salts at the dialysis step, and by the difference in equivalent weights of t he salts present before and after ashing, (For footnotes (a ) to (d) see Table 2).

COMPOSITION OF SPERM WHALE MYOGLOBIN TABLE

675

7

Amino acid analyses of hydrolysates of myoglobin component V Grams amino acid per 100 g protein

Amino acid

Amino acid Number residues Calculated of Average per 100 g number of residues Time of hydrolysis protein residues to nearest 24hr 72hr integer g

Aspartic acid 5·21 Glutamic acid 13·6 Glycine 4·06 Alanine 7·22 Valine (4-44) Leucine 11·7 Isoleucine (5'51) 2·72 Serine Threonine 2·83 Methionine 1·45° Proline 2·22 Phenylalanine 4·95 Tyrosine 2·68 d 9·44 Histidine Lysine 14·1 Arginine 3·68 Tryptophan Amide ammonia 0·69 TotaI!

5·30 13·8 4·14 7·51 4·66 12·1 5·93 a 2·24 2·56 1·48° 2·38 5·09 2·57 d 8·77 13·6 3·32 0·80

5·26 13·7 4·10 7·37 4·66 11·9 5·93 a 3·00 b 2'98b 1·47° 2·30 5·02 2·63 d 9·10 13·9 3'50 2·22 k 0'63f

4·55 12·0 3·12 6·88 3·94 10·3 5·12 2·48 2·53 1·29 1·94 4·47 2·37 8·04 12·2 3·14 2·02

99·7

86·4

7·95 18·8 11·0 16·6 8·01 18·2 9·09 5·74 5·03 1·98 4·03 6·12 2·92 11·8 19·1 4·05 2·19 7·4

8 19 11 17 8 18 9 6 5 2 4 6 3 12 19 4 2 (7) 153

Values are calculated for the anhydrous, ash-free protein. The averages do not include the values in parentheses. The tryptophan value represents one independent determination. totals do not include the values for the heme group. The low recoveries were occasioned by incomplete removal of salts at the dialysis step, and the difference in equivalent weights of the salts present before and after ashing. (For footnotes (a) to (d) and (j) see Table 2). k

1 The

(e) Dinitrophenylation of myoglobin

On the basis of the composition shown in Table 2 myoglobin would be expected on complete dinitrophenylation to yield a derivative containing 35 dinitrophenyl groups. Moreover, if the protons removed from the molecule in titration from pH 5·5 to pH 9 are included, complete dinitrophenylation should be accompanied by the liberation of 56 protons per mole: 38 protons from s-amino groups, 2 protons from the o-amino group, 12 protons from the imidazole side-chains, 3 protons from the phenolic sidechains, and one proton from "acid group III" (associated with the loss of a proton from the water molecule linked to the ferric iron of the heme group in ferrimyoglobin). Separate experiments with native protein and protein denatured by exposure to a pH of3 have shown that approximately 10 equivalents of base are consumed when the pH ofthe protein solution is raised from 7·6 to 9·0. A typical pH-stat trace obtained during the dinitrophenylation of myoglobins IV + V is shown in Fig. 3. In the experiment shown the uptake of base as a consequence of dinitrophenylation was found to be 35 equivalents per mole of protein. The total base consumption was 45 equivalents per mole, 80% of the expected quantity. When an acid-denatured sample of myoglobins IV + V was similarly dinitrophenylated the total base uptake was 90% of that expected.

676

A. B. EDMUNDSON AND C. H. W. HIRS

In comparing these titration values it must be noted that the consumption of base due to substitution reactions is measured by extrapolation of the zero order end-slope of the pH-stat trace to time zero. This method is only satisfactory for the quantitative estimation of substitution when the substitution reactions proceed very much more rapidly than the hydrolysis of the reagent by water. Those groups reacting with fluorodinitrobenzene at rates comparable to the rate at which the reagent is hydrolysed will contribute an essentially asymptotic component to the end-slope, while substitutions taking place at a slower rate than the hydrolysis are not capable of detection at all. The less than theoretical uptake of base during the dinitrophenylation therefore reflects the fact that some of the substitutions in myoglobin were progressing at a relatively low rate, and the possibility of "shielding" of certain residues is suggested by the enhancement in dinitrophenylation that was measured after the protein had been denatured. 20·0 18'0 I

I

0

16·0

'0 Vl

+-' c; ~

14'0

0

>

':; o:

12'0

ClI

eu

i:

10

20 30 40 Time (min)

50

60

FIG. 3. Dinitrophenylation of myoglobin. A tracing of the alkali consumption with time, as registered by a pfl-stat, is shown. Initial volume, 3·0 ml. of an aqueous solution of 0·242 p.moles of myoglobins IV + V. The titration of the solution from pH 7·6 to 9,0, the pH at which dinitrophenylation was performed, is not shown. Temperature, 40°C. The buret contained 0·12 N-NaOH; 0·1 rnl. of Lfiuoro-z.d-dinitrobeneene was used.

Qualitative identification of the dinitrophenylamino acids in the ether-soluble fraction from acid hydrolysates of dinitrophenylated myoglobin by paper chromatography revealed the most prominent spots moving with R F values of 0,36, 0,56, 0·71 and 0·84 in the tert-amyl alcohol, phthalate buffer solvent system. The first three of these spots were identified as s-dinit.rophenyllyaine, dinitrophenol and dinitrophenylvaline, respectively. The spot migrating with an R F of 0·84 was not identified and may have been an artefact; it moved close to the position (R F = 0,88) reported (Blackburn & Lowther, 1951) for dinitrophenylleucine. The principal products found in alkaline hydrolysates of the ether-soluble fraction were valine and tyrosine. For example, after alkaline hydrolysis of the ether-soluble fraction from a 48-hour acid hydrolysate of dinitrophenylated myoglobin quantitative amino acid analysis demonstrated the presence of valine, tyrosine, glutamic acid and serine in over-all yields of 48, 17, 8 and 6%, respectively, of one residue per mole (analyses for basic amino acids were not performed).

COMPOSITION OF SPERM WHALE MYOGLOBIN

677

The results obtained upon quantitative amino acid analysis of the aqueous fraction from acid hydrolysates of dinitrophenylated myoglobin are presented in Table 8. The presence of free tyrosine, histidine, and lysine in the hydrolysates of the dinitrophenylated protein is significant. Acid hydrolysis does not cleave the dinitrophenylsubstitution products of these amino acids to re-form the parent amino acids (Hirs, Halmann & Kycia, 1961). The presence of tyrosine, histidine and lysine in the hydrolysates therefore demonstrates that complete dinitrophenylation was not attained by TABLE

8

Amino acid analyses of dinitrophenylated myoglobin components IV + V Myoglobin Dinitrophenylated components myoglobin IV + V Time of hydrolysis (hr) 48 24 48

Amino acid

Molar ratios of constituent amino acids Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Methionine Proline Phenylalanine Tyrosine Histidine Lysine Arginine

7·90 19·4 1l·0 17·2 7·93 17·8 8·83&

3·90 4·20 1·94 c 4·09 6·26 2·93 d 1l·9 19·2 3·91

8·05 19·3

7·89 19·1

IH

10·8

17·0 6·65 17·7 8·04& 5·01 4·70 1·73 c 4·11 5·88 0·07 d 0·43 0·62 4·12

17·0 6·85 18·0 8·74& 4·02 4·34 1·52 c 3·98 5·92 0·07 d 0·52 0·61 4·05

Reaction with I-fluoro-2,4-dinitrobenzene took place in aqueous solution at pH 9·0 and at 40°C; dinitrophenylated myoglobin was produced from 1·60/Lmoles of components IV + V in 90 min. For further details, see the text. After the dinitrophenylated proteins had been subjected to acid hydrolysis at 1l0°C, the dinitrophenylamino acids were extracted with ether, and a sample of the extracted hydrolysate was taken for amino acid analysis. The values for serine and threonine are uncorrected values. (For footnotes see Table 2.)

exposure of myoglobin at pH 9 and at 40°C to the action of fluorodinitrobenzene for 90 minutes. The residual quantities of tyrosine, histidine, and lysine (cf. Table 8) show that, statistically, the reaction was 96% complete. This value should be contrasted with the lower estimates arrived at by means of the pH-stat measurements. Table 8 shows that approximately one residue less of valine was present per mole in hydrolysates of the dinitrophenylated protein than in a control hydrolysate of the myoglobin used in the experiment. The significance of the presence of serine and glutamic acid in the ether-soluble fraction from hydrolysates of dinitrophenylated myoglobin must be considered. Since serine is largely destroyed by alkaline hydrolysis and dinitrophenylserine is notably unstable on acid hydrolysis, the presence of even traces of serine as the dinitrophenyl derivative in the ether-soluble fraction could imply the presence of a serine end-group in myoglobin. The analyses in Table 8,

678

A. B. EDMUNDSON AND C. H. W. HIRS

however, do not reveal differences between the serine content of hydrolysates of myoglobin and dinitrophenylated myoglobin. The presence of approximately onetenth residue equivalent of free serine in the barium hydroxide hydrolysate of the ether-soluble fraction was therefore due to some procedural artefact introduced during the alkaline hydrolysis. The precision attainable in the quantitative methods used does not permit difference analysis for glutamic acid in myoglobin. In contrast to serine, however, glutamic acid is relatively stable on acid and alkaline hydrolysis, and the presence of one-twelfth of a residue equivalent of glutamic acid in the alkaline hydrolysate of the ether-soluble fraction from dinitrophenylated myoglobin undoubtedly represents the contribution of an authentic end-group. In view of the relatively low yield obtained it is possible that the glutamic acid end-group may be due to the presence of myoglobin molecules that have undergone cleavage at an unusually sensitive peptide bond, either during the procedures associated with end-group determination or in the isolation of the protein from the effiuent of the IRe-50 columns. The presence of tyrosine in the hydrolysate of the ether-soluble dinitrophenylamino acids may be ascribed to the presence of o-dinitrophenyltyrosine. The only amino-terminal amino acid revealed to be present in significant quantities by examination of the ether-soluble fraction and by difference analysis thus was valine. By qualitative methods Ingram (1956) had previously examined the aminoterminal end-groups in crystalline sperm whale myoglobin, and had reached the conclusion that valine is the only N-terminal amino acid in the protein. (f) The action of carboxypeptidase on myoglobin

As will be shown in the paper that follows (Edmundson & Hirs, 1962a) all but one of the peptides obtained upon tryptic hydrolysis of myoglobin contain lysine or arginine. This would be the expected result if the myoglobin molecule consisted of a single polypeptide chain and the specificity of trypsin were rigorously maintained. Since the results presented in the previous section indicate that myoglobin possesses only one ex-amino group, the peptide devoid of lysine or arginine in the tryptic hydrolysates may be assumed to derive from the carboxyl end of the chain. In this event, on the basis of the assumptions just given, and provided the carboxyl end of the chain is not buried in the tertiary structure, both myoglobin and the peptide derived from the carboxyl terminus should to a first approximation exhibit similar behavior upon attack by carboxypeptidase. It will be shown in an accompanying paper (Edmundson & Hirs, 1962b) that the carboxyl-terminal peptide has the sequence: Glu-Leu-Gly-Tyr-Gly-Glu(NH 2 ) Table 9 shows that the extent of hydrolysis under the conditions used of the glycylglutamine, tyrosylglycine and glycyltyrosine bonds by carboxypeptidase at pH 8 is very nearly the samej, It is possible that this result is obtained because the ratelimiting step in the hydrolysis is the removal of the glutamine residue. It is also apparent that after the removal of three residues the residual tripeptide is attacked comparatively slowly at the leucyl-glycine bond. The results obtained with the peptide are paralleled by the results obtained with myoglobin and the globin derived from it. As Table 9 shows, the amino acids split

t The analyses for glutamine do not carry corrections for the extent of conversion of glutamine to pyrrolidone carboxylate during the enzymic hydrolysis.

COMPOSITION OF SPERM WHALE MYOGLOBIN

679

from the proteins in the most significant quantities were glutamine, glycine and tyrosine. The most notable difference in the results obtained with myoglobin and globin is in the tyrosine values, which show that tyrosine was more completely removed from globin than from myoglobin under comparable conditions. Possibly this difference of behavior resulted because the globin (as prepared in this work) was more extensively denatured than the parent protein. Unlike the peptide, the intact protein on attack by carboxypeptidase formed leucine in a relatively constant ratio to the quantity of TABLE

9

Amino acids liberated from peptide Tryp-16 (cf. Edmundson &: Hirs, (1962b», globin derived from myoglobin components I V + V, and myoglobin components IV + V by the action of carboxypeptidase Composition of 'I'ryp-Hl Time of hydrolysis (min) Enzyme concn. /Lg protein N /m1. Amino acid Glutamic acid } Glutamine Glycine Leucine Tyrosine Aspartic acid Alanine Isoleucine Threonine Yield in per cent

Su bstrate Tryp-16 Globin lot 1

Myoglobin

Globin lot 2

60

15

30

120

143

162

162

239

Molar ratios of constituent amino acids 1·96 2·04 1·00 0·96

0·01 0·84 1·07 1·00

52

0·10 0·93 1-13 0·24 1·00

0·06 1·00 1-12 0·20 0·36

0·21

0·33 0·10

16

11

0·11 0·83

HO 0·28 1·00 0·02 0·12 0·06 0·04 53

glutamine and glycine removed. This observation suggests that in the protein, cleavage of the leucyl-glycine bond proceeds more rapidly than it does in the peptide. Failure to obtain leucin e and glutamic acid from t he peptide may be attributed to t he resistance of dipeptides to the action of carboxypeptidase. The presence of glutamic acid in the reaction product s from the protein probably indicat es that the enzyme was att acking the bond between glutamic acid and the basic amino acid t hat must pre cede glu t amic acid in the amin o acid sequence. Ot her amino acids, not ably alanine, were also present in t he reaction products from the proteins; but the quantities of these other amino acids decreased relative t o the quantities in which the principal amino acids were liberated as the extent of hydrolysis increased. It is therefore possible that these relatively constant quantities of other amino acids were formed because carboxypeptidase attacked free terminal groups previously introduced into the protein as a consequence of some step(s) in the isolation procedure. In the previous section attention was drawn to similar observations that suggested the presence of a fractional amount of an amino-terminal glutamic acid group in the protein as prepared in this work.

680

A. B. EDMUNDSON AND C. H. W. HIRS

4. Discussion (a) The components of crystalline sperm whale myoglobin

The pH range in which equilibrium chromatography of sperm whale myoglobin on the sodium form of IRO-50 may be attained is narrow. At 4°0 in sodium citratechloride buffer, 0'34:N in sodium ion, the range is between pH 5·82 and pH 5·92 (as measured at 25°0), a range similar to those found in the same buffer by Boardman & Partridge (1955) for the chromatography of bovine adult ferri-hemoglobin and carboxyhemoglobin, by Boardman & Adair (1956) for the chromatography of equine heart myoglobin, and by Timmer, van der Helm & Huisman (1957) for the chromatography of fetal and adult bovine myoglobin. The pronounced selectivity of the conditions implies that similarities exist in the way the citrate complexes of these related but different heme proteins interact with the surface of the resin. The tailing of the myoglobin zones on the chromatograms, undoubtedly accentuated in part by the necessity to work at a temperature low enough to prevent denaturation, makes it impossible to attain a clear-cut separation of the individual components. Extensive tailing of the zones also may serve to mask the presence of minor components in the effiuent pattern in those regions of the chromatogram between major zones. Moreover, the narrow range of pH in which equilibrium chromatography is possible precludes rechromatography under different conditions on the same resin for the purpose of demonstrating chromatographic homogeneity. These considerations underline the necessity for caution in interpreting the chromatographic results presented in this paper. At best it may be stated that there must be at least five different molecular species present in crystalline sperm whale myoglobin. The difference in chromatographic behavior of the components of the crystalline protein is at present without explanation. If, as appears likely in view of the identity of their amino acid compositions, all the components are present in the different crystalline forms of myoglobin, the X-ray results demonstrate that they are capable of assuming the same conformation in the crystal lattice and, by inference, must possess closely similar conformations when in solution. The ease with which myoglobin denatures argues in favor of the view that relatively small fluctuations in conformation of the molecule are irreversible and cannot be tolerated. On this basis the difference in chromatographic behavior of the components of myoglobin is unlikely to be due to the existence of independent stable conformational variants of the same basic structural skeleton in solution. In this regard it is significant that the crystallographically identical myoglobin of seals (Scouloudi, 1960) has been resolved into five components by chromatography on OM-cellulose by Rumen (1959, 1960) who has demonstrated that each of the components is electrophoretically homogeneous but of different mobility. This result strongly suggests that the separations on OM-cellulose of the seal myoglobin components are at least in part due to charge differences. The possibility that such charge differences also exist among the components of sperm whale myoglobin is presently under investigation. Schmid (1949) demonstrated many years ago that finback-whale myoglobin is electrophoretically inhomogeneous. Even if the binding of sperm whale myoglobin citrate complex by IRO·50 is primarily due to interaction of a "preferred" binding site on the protein with the resin surface, a charge difference in a part of the molecule located more remote from the resin surface would be expected to be capable of exerting a significant effect on the binding.

COMPO SI T ION OF S P E R M WHALE MYO GLOBIN

681

(b) A sp ects of the gross structure of sperm whale myoglobin

The X -ray crystallographic studies have provided dir ect proof t hat the molecule contains a single polyp eptide chai n . The end-group analyses presented in t his pap er were interpreted in similar t erm s at a time when the definitive evidence of the 2 A Fourier projections was not yet available. In the light of t he most recent knowledge, the results obtain ed by chemical methods wit h myoglobin are of interest in the more general context of t he influence of pr ot ein conform ation on the accessibility of the end-groups and certain side -chains to reagents and enzymes. Th e X-ray crystallographic st udies show t hat the carboxyl -terminal end of the molecule is pr esent as a non-helical segment (Kendrew et al., 1961) of five to six residu es, of which the last t wo in the chain, glycine and glutamine, are in a relati vely " accessible" location on the extern al "s urface" of the molecule. The next residue in line, tyrosine, is apparently not hydrogen-bonded by it s phenolic hydroxyl group t o any group located on an adj acent helix formed by a remote segment of the peptide chain. Thes~ three residues are removed from myoglobin in the native configuration (at pH 8) by carboxypeptidase at rates at least comp arable to the rates observed when the carboxyl terminal peptide from the protein is exposed to the enzyme. It would thus appear that any hyd rogen bond capable of being formed by the tyrosine residu e at the carboxyl end of the chain is either ab sent in solut ion, or, if present, is sufficiently weak t o permi t ready dissociation of t he two members connect ed by it. Th e results obtained on dinit ropheny lation of myoglobin indicat e that all the e-amino, imidazole and phenolic side-chains become accessible t o fluorodinitrobenzene in the course of the reacti on . It may be presumed that the initial rections take place at the "surface" of the molecule. The introduction of the relat ively non-polar dinitrophe nyl groups at locations pr eviously occupied by charged (and undoubtedly hydrated) f-amino, imidazole or phenolic side -chains would serve to disturb t he equilibrium between those second ary forces du e to polar bonding and those due to non-polar bonding in the molecule, and t hus lead to progressive unfolding as more such groups were introduced. The alm ost instantaneous appearance of insoluble material after initiation of t he reaction between myoglobin and fluorodinitrobenzene suggests that denaturation takes place very early in t he pro cess, possibly with the introduction of at most one or t wo dinitrophenyl groups. Thereafter reaction continues in a het erogeneous medium betw een t he precipitated protein and the reagent in solut ion . The fact that virtually complet e sub stitution may be achi eved under such conditions implies that the denatured protein mu st be formed essentially in a single step before it separates from solution as a pr ecipitate. It thus appear s that the introduction of the first dinitrophenyl groups at side-chains on the "s urface" of the molecule may cau se a complete unfolding of the t ertiary structure and the assumption of the fully denatured state. Since the X -ray sect ions at a resolut ion of 2 A reveal the aminot erminal valine residue in crystalline myoglobin t o be in an "accessible" location, it would be expe ct ed that the a-amino group sh ould be the most rea ctive of the functional groups t owards fluor odinitrobenzene (at pH 9) on t he "surfa ce" of the molecule, and t hat the introdu cti on of a dinit roph enyl group in this region of the molecule would be one of the principal features of t he denaturing pro cess. This work was initiated at The Rockefeller Ins titute in the laboratories of Dr. S. Moore and Dr. W. H. Stein, t o whom we are indebte d for advice and encouragement . Pr eliminary reports of this work taken in par t from a Thesis submitted by A. B. Edmundson in par tial

682

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fulfilment of the requirements of the Degree of Doctor of Philosophy at The Rockefeller Institute have already appeared (Edmundson & Hirs, 1959, 1961). His work at Cambridge has been facilitated by grants from the United States Public Health Service for a fellowship and for provision of an automatic amino acid analyser (grant nos. A-3032 and A-3032(SI)). Research at the Brookhaven National Laboratory was performed under the auspices of the United States Atomic Energy Commission. We wish to thank Dr. J. C. Kendrew for his continued support of this research and Mrs. Margaret Ross for her skilled assistance. REFERENCES Blackburn, S. & Lowther, A. G. (1951). Biochem, J. 48, 126. Boardman, N. K. & Adair, G. S. (1956). Nature, 177, 1078. Boardman, N. K. & Partridge, S. M. (1955). Biochem, J. 59, 543. Braunitzer, G., Gehring-Muller, R., Hilschmann, N., Hilse, K., Hobom, G., Rudloff, V. & Wittmann-Liebold, B. (1961). Z. physiol. Ohem, 325, 283. Crampton, C. F., Moore, S. & Stein, W. H. (1955). J. Biol. Ohem. 215, 787. Dreze, A. (1956). Biochem, J. 62, 3P. Edmundson, A. B. & Hirs, C. H. W. (1959). Fed. Proc. 18, 220. Edmundson, A. B. & Hirs, C. H. W. (1961). Nature, 190, 663. Edmundson, A. B. & Hirs, C. H. W. (1962a). J. Mol. Biol. 5, 683. Edmundson, A. B. & Hirs, C. H. W. (1962b). J. Mol. Biol. 5, 706. Hamilton, P. B. (1958). Analyt. Ohern; 30, 914. Hirs, C. H. W. (1955). In Methods in Enzymology, ed. by S. P. Colowick & N. O. Kaplan, vol. I, p. 113. New York: Academic Press. Hirs, C. H. W., Halmann, M. & Kycia, J. H. (1961). In Biological Structure and Function, ed. by T. W. Goodwin & O. Lindberg, vol, I, p. 41. New York: Academic Press. Hirs, C. H. W., Moore, S. & Stein, W. H. (1960). J. Biol. Ohem, 235, 633. Hirs, C. H. W., Stein, W. H. & Moore, S. (1954). J. Biol. Ohern; 211, 941. Ingram, V. M. (1956). As quoted in Kendrew, J. C. & Parrish, R. G. Proc. Roy. Soc. A, 238,305. Kendrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H. & Phillips, D. C. (1958). Nature, 181, 662. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. & Shore, V. C. (1960). Nature, 185, 422. Kendrew, J. C. & Parrish, R. G. (1956). Proc, Roy. Soc. A, 238, 305. Kendrew, J. C., Parrish, R. G., Marrack, J. R. & Orlans, E. S. (1954). Nature, 174, 946. Kendrew, J. C., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, D. C. & Shore, V. C. (1961). Nature, 190, 666. King, T. P. & Craig, L. C. (1955). J. Amer. Ohem. Soc. 77, 6624. Konigsberg, W., Guidotti, G. & Hill, R. J. (1961). J. Biol. Ohem. 236, PC 55. Levy, A. L. (1954). Nature, 174, 126. Levy, A. L., Geschwind, I. 1. & Li, C. H. (1955). J. Biol. Ohem, 213, 187. Mills, G. L. (1952). Biochem, J. 50, 707. Moore, S., Spackman, D. H. & Stein, W. H. (1958). Analyt. Ohem, 30, 1185. Moore, S. & Stein, W. H. (1949). J. Biol. Chern: 178, 53. Perutz, M. F., Rossmann, M. G., Cullis, A. F., Muirhead, H., Will, G. & North, A. C. T. (1960). Nature, 185, 416. Rumen, N. M. (1959). Acta chem, Scand. 13, 1542. Rumen, N. M. (1960). Acta chem, Scand. 14, 1217. Schmid, K. (1949). Helv. chim. acta. 32, 105. Scouloudi, H. (1960). Proc. Roy. Soc. A, 258, 181. Spackman, D. H., Stein, W. H. & Moore, S. (1958). Analyt. Ohem. 30, 1190. Theorell, H. & Akeson, A. (1955). Ann. Acad. Sci. Fenn., 2A, 60, 303. Timmer, R., van der Helm, H. J. & Huisman, T. H. J. (1957). Nature, 180, 239.