Comp. Biochem. Physiol., 1976, Vol. 54B, pp. 253 to 259. Peroamon Press. Printed in Great Britain

CHARACTERIZATION OF MYOGLOBINS FROM ATLANTIC AND PACIFIC GREEN SEA TURTLES JOHN D. WILLIAMS, JR. AND W. DUANE BROWN

Institute of Marine Resources, University of California, Davis, CA 95616, U.S.A. (Received 23 June 1975) Abstract--1. This paper presents information on the isolation, purification, and characterization of myoglobins from two sub-species of green sea turtles, Chelonia mydas mydas (Atlantic) and Chelonia mydas caranigra (Pacific). 2. Data presented include amino acid composition, extinction coefficients for a variety of derivatives, estimation of molecular weight by Sephadex chromatography and acrylamide gel electrophoresis, isoelectric points, extent of heterogeneity, and oxygen equilibria. 3. Also included is a picture of a cross-section of muscle fibers stained for myoglobin.

INTRODUCTION

FOLLOWING the elucidation of primary and three-dimensional structure of sperm whale myoglobin (Mb) (Edmundson, 1965; Kendrew et al., 1960) there have appeared a number of reports detailing characteristics of myoglobins from other sources. Myoglobins studied for the most part have been those of vertebrate animals, including aquatic mammals other than sperm whale (Rumen, 1960; Atassi & Saplan, 1966; Hapner et al. 1968; Hartzell et al., 1968; Bradshaw et al., 1969; Vigna et al., 1974); man (Perkoff et al., 1962; Hill et al., 1969; Herrera & Lehmann, 1971b); gibbon (Herrera & Lehmann, 1971a); dog (Dautrevaux et al., 1971); horse (Dautrevaux et al., 1969); cattle (Quinn & Pearson, 1964; Satterlee & Snyder, 1969; Han et al., 1970); sheep (Satterlee & Zachariah, 1972; Han et al., 1972); pig (Nakanishi & Isumimoto, 1972; Satterlee & Zachariah, 1972; Floc'h et al., 1973); camel (Awad & Kotite, 1966); badger (Tetaert et al., 1973); kangaroo (Air & Thompson, 1971); chicken (Goldbloom & Brown, 1971; Deconinck et al., 1972); and tuna (Matsuura & Hashimoto, 1955; Konosu et al., 1958; Brown et al., 1962; Hirs & O1cott, 1964). Information from the reports just cited has been valuable for investigators interested in evaluating relationships between structure and reactivity of these proteins. The potential for such evaluation should be enhanced by the availability of data on other myoglobins. To that end we present herein a description of the isolation, purification and characterization of myoglobins from two species of green sea turtles, CheIonia mydas mydas (Atlantic) and Chelonia mydas caraniora (Pacific). These reptiles are noted for their light-colored muscle and deep, prolonged diving. Dives may exceed 100 m in depth for periods up to 5 hr (Berkson, 1966). Their heart beat has been demonstrated to slow to as little as one beat per 9 min (Berkson, 1967). These turtles are incapable of dermally absorbing oxygen, but show a marked capacity to survive by anaerobiosis (30). We are aware of only one other report dealing in part with sea turtle myoglobin; it includes some data ('.B.P.(B) 54/2B D

253

on the properties of myoglobin from Caretta caretta (Tomita & Tsuchiya, 1971). MATERIALS AND METHODS

Materials Marineworld, Africa U.S.A. (Redwood City, Calif.) supplied a female Pacific green sea turtle weighing approx. 80 kg. Arrangements were made in Florida for the purchase and shipment of a live female Atlantic green sea turtle weighing 30 kg. Both turtle specimens were stunned and decapitated. The carapace was removed and the skeletal muscles collected. The muscles were frozen in plastic bags at -10°C until ready for further use. Ordinary chemicals used were reagent grade or better; sources of other materials are indicated when their use is described in the following paragraphs. Methods Within minutes after the specimens were sacrificed, small center blocks of muscle (2-3 mm) were excised and prepared for histochemical staining as described by James (1968). Sections were quick frozen with Cryokwik (International Equipment Co.) or an isopentane/dry ice bath and sliced into transverse sections varying in thickness from 12 to 50/ma. For isolation and purification of myoglobin, muscle was freed from as much fat and connective tissue as possible. A portion of muscle was blended with 1.5 vol cold distilled water in a Waring blender for 2 min. The resultant slurry was centrifuged at 7700 g in a refrigerated centrifuge to remove insoluble matter. The supernatant was decanted and dialyzed (12°C) against ammonium sulfate solutions adjusted to pH 7"0 with Trizma base (Sigma) such that protein precipitates were collected at 10% increments of saturation from 50 to 100% saturation. Ammonium sulfate pastes which contained significant amounts of myoglobin were dissolved in a minimum amount of water and desalted by passing over a 2"5 x 30 can column of Sephadex G-25 (Pharmacia). The salt-free solution was then passed through a 2'5 x 90 cm column of Sephadex G-75 (Pharmacia), regular mesh equilibrated with 0.05 M Tris-HCl, pH 8.0. Flow rate was maintained at 20 ml/hr with an LKB 10020 Perpex peristaltic pump. Occasionally, further purification was done by use of CM-sephadex C-50 (Pharmacia). Column size was 2-5 x 60 crn. Elution was with 0-05

254

JOHN D. WILLIAMS~JR. AND W. DUANE BROWN

M Tri~HCI, pH 6.3. Flow rate was maintained at 17 ml/hr using a peristalic pump. Yellowfin tuna myoglobin was prepared in a similar fashion using fish supplied by Star-Kist Foods. Sperm whale myoglobin was from Miles-Seravac and was purified by Sephadex G-75 chromatography. Purity was determined by two polyacrylamide gel electrophoresis systems, one with polyacrylamide charge gels using the general system described by Davis (1964) and the other with a gel system containing sodium dodecyl sulfate (SDS) (Sigma), as described by Weber & Osborn (1969). Spectra were recorded from 700 to 250 nm on a Cary model 15 recording spectrophotometer. Complete spectra of metMb, MbO2, Mb, MbCN, and MbCO were determined for the Pacific turtle myoglobin, and a metMb Soret extinction coefficient was determined for Atlantic turtle myoglobin. Complete oxidation to metMb was achieved with potassium ferricyanide, the excess of which was removed with mixed bed resin (Bio-Rad, AG 501-X8). Dilutions of 1:5 were made with 0-05 M Tris-HCl, pH 7.0. Samples scanned in the Soret region were diluted 1:20. Cyanometmyoglobin was formed by addition of a 2-fold M excess of potassium cyanide. Removal of excess reagent and dilution patterns were as previously described for metMb. Carboxymyoglobin was formed by first converting the myoglobin to the ferrous form by reduction with sodium dithionite. A few grains of dithionite were dissolved in a solution of myoglobin and quickly removed by addition of mixed bed resin as described. Carbon monoxide (Matheson) was bubbled through the ferromyoglobin for 4~5 rain. Dilution was as previously described. Spectra of Mb and MbO2 were determined on the same samples. A few grains of sodium dithionite were added to a myoglobin solution and quickly removed by immediately passing the sample over a column of mixed bed resin (0.5 x 5 cm) at 12C. Deoxy myoglobin was formed by placing the MbO2 sample plus 3 ml of 0"01 M phosphate buffer, pH 7-1, in a 30 ml vacuum flask. The solution was packed in ice on a shaking water bath and purified Nz (LiquidCarbonic "High-Pure") was bubbled through the solution for 3 hr. The effluent N2 passed through a Thunberg cuvette. At the end of 3 hr the myoglobin solution was transferred into the cuvette and the cuvette was sealed. After recording the spectra of Mb the Thunberg cuvette was opened and oxygen was bubbled through the solution. The sample was then scanned as before and the Mbg2 spectra recorded. The concentration of myoglobin in the above samples was determined by addition of 1 crystal of potassium ferricyanide and measurement of the resultant Sorer absorption. Prior to spectral studies concentrations of purified myoglobin solutions had been determined by use of atomic absorption spectroscopy. Standard solutions of 5, 10, 15 and 20 ppm iron were prepared, using electrolytic iron (Matheson, Coleman and Bell) that had been dried under vacuum. All determinations were made using a Perkin Elmer model 303 Atomic Absorption Spectrometer with a hollow cathode Fe lamp and assuming the Fe content of the myoglobins to be 0-33%. At the completion of each iron analysis, an aliquot of myoglobin (less than 1 rag) was treated with 1 pl of 5~o K3Fe[CN)6 and the Soret absorption for metMb determined. For infrared analyses of heme, Pacific turtle and sperm whale myoglobins were made to contain 0.1 N HCI and were extracted repeatedly with 2-butanone. The hemes contained in the 2-butanone were freed of the solvent by nitrogen purge and dissolved in acetone. The solutions were applied to both sides of an Irtran 2 (ZnS) crystal contained in an attenuated total reflectance unit (Barnes Engineering Co., Stanford, Conn.) and the acetone allowed to evaporate. The unit was then mounted on a Beckman IR 8 Spectrometer and scans of the two heine extracts recorded.

A variation of the procedure of Whitaker 119631 wa~ used for molecular weight determination by gel filtration (36), using Sephadex G-75 Superfine Imesh. l(g40 in. Column size was 1.5 x 90 cm. An ascending flow rate of 4 ml/hr of 0.05 M Tris-HCl, pH 8.0 was used. Elution was monitored with a LKB Uvicord I1 at 280 nm. Standard proteins were chymotrypsinogen (Pharmacia). sperm whale myoglobin (Miles-Seravac), and ribonuclease A (Pharmaciat. Partition coefficients were determined by K,~ = ( 1 ~ - VoL,(~, -- 1,~ where V~, is the elution volume of the protein, ~ the void volume of the column, and I'~ the total volume of the column. SDS-acrylamide gels were used as a second method of estimating molecular weight. Gels were 10% acrylamide and cast at 8 cm lengths. Standard proteins used were. from Pharmacia: ovalbumin, aldolase, ribonuclease, and chymotrypsinogen; from Sigma: egg white lysozyme, carbonic anhydrase, and 7-globulin (Cohn fraction II); and from Miles-Seravac: sperm whale myoglobin. Chymotrypsinogen was included as a reference protein in each gel. The method of Dunker & Rueckert (1969) was used to determine mobility and to establish a standard curve. To test the influence of charge variations on the relative mobilities of sperm whale and sea turtle myoglobins, the myoglobins were treated with maleic anhydride at a 100-fold molar excess over amino groups by modification of the methodology described by Butler et al. (1969). Both myoglobins were buffered in 1 M N-ethyl morpholine buffer, pH 8'5. Maleic anhydride was added as a dr>' powder by three additions over a period of 30 min. The maleyated proteins were run on SDS-polyacrylamide gels as described above. For amino acid analyses protein samples were hydrolyzed in constant boiling 6 N HC1 in sealed, evacuated tubes for periods of 24 and 72 hr in a 108"C oil bath. Hydrolysates were evaporated to dryness and chromatography was done on a 0.63 x 66 cm column of Aminex A-5 (Bio--Rad) using the general procedure of Moore, Spackman and Stein (Moore et al., t958; Spackman et al., 1958) modified for a one column system using a Technicon analytical system. Estimation of amino acid content of both the Atlantic and Pacific turtle myoglobins was made assuming 153 residues per molecule. Values for serine and threonine were corrected by interpolating to zero time. Cysteine values were determined by performic acid oxidation as described by Hirs (1967). Ellman's (1959) reagent and p-chloromurcuribenzoate (Sigma) were used in attempts to demonstrate the presence of a free sulffiydryl. Tryptophan was determined spectrophotometrically by the general methodology of Edelhoch (1967). Sperm whale myoglobin was analyzed concurrently with samples of turtle myoglobins. Electrofocusing studies were conducted using acrylamide gels as described by Rice & Horst (1972l. 75?0 acrylamide gels were cast from a solution containing in 20 mt: 1-5 g acrylamide, 40 mg bis-acryl-amide, 20 Id N , N , N ' , N ' - t e t r a methylene diamine, 5 mg ammonium persulfate, and 1 ml of 400,/, ampholytc (LKB), pH 3-10. Determinations were made using myoglobins from both turtles, yellowfin tuna and sperm whale. Samples of Pacific turtle myoglobin were run after Sephadex G-75 chromatography and later after CM-Sephadex purification. Atlantic turtle myoglobin was run only after both steps of purification. Sperm whale and yellowfin tuna myoglobin were purified on CM-Sephadex. After completion of each run, the gels were treated in 1 of 3 manners: (a) To determine pH gradients, parallel blank gels were frozen and sliced into 2 mm lengths with a gel slicer (Hoefer Scientific Instruments). Each slice was immersed in 2 ml distilled water. After 2 hr, the pH of each was recorded and a plot of the pH gradient constructed. (b) For determination of pl values the visible

Characterization of myoglobins

255

Fig. l. Photomicrograph of section of claviculo brachialis muscle of Atlantic sea turtle; 16x magnification; stained for myoglobin, which shows as darker color. major band of some gels was excised with a razor blade and immersed in 2 ml distilled water. The pH of the band was then measured directly. (c) For further evaluation and determination of microheterogeneity, electrofocusing gels were removed from the gel tubes and stained for myoglobin by immersion in a freshly prepared solution containing in 100 ml: 100 mg benzidine, 70 ml of 95~o ethanol, l0 ml of 1"5 M sodium acetate buffer, pH 4.7, and 2 ml of 30~ hydrogen peroxide. After 15 min the gels were washed in three changes of distilled water and stored in a solution of 5~o methanol, 7"5~o acetic acid. The stained gels were scanned at 409 nm on a Gilford model 2400 Recording Spectrophotometer. Oxygen equilibrium studies were conducted using a tonometer designed after that developed by Benesch et al. (1965) and manufactured by Eck and Krebs. A modified procedure of Rossi-Fanelli & Antonini (1958) was followed. Turtle MbO2 was prepared as described above. The MbO2 eluting from the mixed bed resin column was diluted to 9 ml with deionized water and added with 6 ml of 0.05 M Tris-HC1 buffer, pH 7'4 to the tonometer. The excess water was added to allow for evaporation. The tonometer was chilled in an ice bucket and a vacuum applied. After 5-10 min the tonometer was mounted horizontally on a shaking water bath and evacuation was continued for 40 rain. The tonometer was sealed and a septum attached through a Swagelok fitting and rubber tube to the glass tube on the tonometer cap. The space within was evacuated by insertion of a needle through the septum and a vacuum applied for 10 min. The tonometer was then mounted in a Cary Spectrophotometer using a specially constructed enlarged cover which facilitated placing the device in the sample chamber. Complete deoxygenation was determined spectrophotometrically. Injections of known amounts of air were made with a Hamilton l ml gas-tight syringe. Equilibrium was hastened by spinning the tonometer in a rotator designed similarly to the one employed by Benesch et al. (1965). Constant temperature (20°C) was maintained by submerging the rotator in a water bath. No more than 3 injections per-sample were made to minimize autoxidation. The samples were scanned from 650-450 nm. After the third injection, the tonometer contents were exposed to air, and the spectra recorded. If the 580/542 nm absorption ratio was less than

1"04,the values were discarded on the assumption that excess autoxidation had occurred.

RESULTS AND DISCUSSION

Histological localization Figure 1 demonstrates staining results obtained on a sample of Atlantic green sea turtle claviculo brachialis muscle. There are obvious intermediate degrees of staining between type I and type II fibers, as reported by James (1968) for other muscles. As suggested by James (ibM.), the several degrees of intermediate staining suggest that the terminology of red and white muscle fiber should be used specifically to designate myoglobin rich and myoglobin poor fibers, and not fiber types.

Spectra Absorption maxima and extinction coefficients were determined assuming a myoglobin molecular weight of 18,000 Daltons. Table 1 lists values determined for Pacific green sea turtle myoglobin. Wavelength maxima and extinction coefficients for various ligand forms of this protein are in good agreement with other myoglobins, suggesting similar positioning and association of the heme to the apoprotein moiety. The Soret absorption maximum for Atlantic turtle metMb was 409 nm and ~mM °409 was calculated to be 157. We presume from the fact that infrared spectra of hemes removed from sperm whale and Pacific turtle were identical that the heine group of the turtle myoglobin is protoheme IX.

Molecular weight Figure 2 shows the straight line relationship between Kay and log molecular weight as determined on a Sephadex G-75 Superfine column. Pacific sea turtle myoglobin exhibited virtually the same elution

256

JOHN D. WILLIAMS.JR. AND W. Dt~AYEBROWN

Table l. Spectral data for various derivatives of Pacific green sea turtle myoglobin

1.6 'XA []

Derivative

Wavelength, mm

Metmyog]obin

633

4,2

504

7.4

409

Oxyl~yogloh i n

35.2

580

13.8

543

34.0

556

113.

280

34.5

577

| 2,5

541

14,7

423

152.

280

33.8

542

l 0.9

424

ll3.

280

34.0

aBased on molecular weight of 18,0OO Daltons

volume as sperm whale myoglobin, emerging just slightly earlier. When the two myoglobins were mixed and applied to the column, no measurable separation was monitored, Molecular weight for the turtle myoglobin as determined by gel filtration thus appears to be about 18,000 or slightly higher. Figure 3 demonstrates the straight line relationship obtained using SDS-acrylamide gels and plotting log molecular weight vs the quotient of standard protein migration. The same quotient determined for turtle myoglobins gave apparent mol. wt of 15,800 Daltons (+5%) for proteins from both Atlantic and Pacific species. After maleylation of Pacific turtle and sperm whale myoglobin, the two migrated to the same quotient value (equivalent to 18,000 mol. wt).

0.45

'XA

0.40 0.35

~B 030 0.25 0.20

015 t

I

I

x0\

1.0

o.8 0.6

EXxF

\

=E

XG \

0.4

I1.9

434

Cyanometmyoglobi n

1.2

0

12.8 120.

280

Carboxymyoglobi n

z taJ

160.

280

417

Deoxymyoglobin

1.4

a

crd, I

I

t

I

4.1 4.2 43 4.4 4.5 4.6 LOG MOLECULAR WEIGHT

Fig. 2. Determination of molecular weight of Pacific sea turtle myoglobin by gel filtration on Sephadex G-75. A, ribonuclease (mol. wt 13,200); B, sperm whale myoglobin (tool. wt 18,000); C, chymotrypsinogen (mol. wt 25,700); I-1, Pacific green sea turtle myoglobin.

0.2 ,

t

J

,

i

,

,

i

l

i

,

,

4.2 4.4 4.6 4.8 5.0 LOG MOLECULARWEIGHT Fig. 3. Determination of molecular weight of turtle myoglobin by SDS-polyacrylamide gel electrophoresis. A, lysozyme (mol. wt 14,300); B, sperm whale myoglobin (mol. wt 18,000): C, carbonic anhydrase (mol. wt 29,000); D, ;,globulin, light chain (mol. wt 23,500); E, aldolase (mol. wt 40,000); F, ovalbumin (mol. wt 45,000); G, )'-globulin, heavy chain (mol. wt 50,000); [], II, Atlantic and Pacific green turtle myoglobins; O, 0, sperm whale and Pacific : green sea turtle myoglobins modified by reaction with maleic anhydride. The anomalous behavior of the turtle myoglobins on SDS-acrylamide gels is similar to that cited for other proteins (Tung & Knight, 1971; Tung & Knight, 1972). Since the increase in molecular weight resulting from the addition of maleyl groups is insignificant when compared to the effect of charge, it would appear that charge effects are not completely eliminated when turtle myoglobins are subjected to electrophoreses in an SDS environment. Amino acid composition

Table 2 lists the values obtained by amino acid analyses. The two turtle myoglobins appear very similar, with possible differences in lysine, histidine, serine, glutamic acid, proline, and glycine residues. Performic acid oxidations and subsequent amino acid analyses gave consistent values for cysteic acid ranging from 0-9 to 1.1 for Pacific turtle myoglobin and 0"5 to 1.I for Atlantic turtle myoglobin. Cysteine residues have been found infrequently in myoglobins, including those of tuna (Konosu et al., 1958; Brown et al., 1962), shark (Tomita & Tsuchiya, 1971), gibbon (Herrera & Lehmann, 1971a) and man (Hill et al., 1969; Herrera & Lehmann, 1971b). Acceptable degrees of reactivity of free sulfhydryl were difficult to obtain with either native or denatured protein. The reaction of p-chloromercuribenzoate was not quantifiable. Several attempts to quantitate the sulfhydryl content with Ellman's reagent did not give yields above 50°/0 There was an obvious correlation between age of the purified protein sample and demonstrable -SH, but the latter was not affected by treatment with mercaptoethanol. The spectroscopic determination of tryptophan yielded values of 1.9 and 1-8 residues per mole for

Characterization of myoglobins Table 2. Amino acid composition of turtle myoglobins Atlantic Sea Turtle

Restdues a

Aspartic A c i d Threonine Serine Glutamic A c i d

Pacific Sea Turtle

NO. of

Amino Acid

NO. of

Oetm.

Reatdues a

5

13.1_+0.59

12.9LH),3g 8.I b

S

7.2 b

6

5

8.3b

5

8.6b

5

5

21.3~I.02

5

Prollne

5.3+0.35

6

6.1_+0.36

5

Glyclne

8.4+0.16

6

9.6_-H),74

5

Alanine

13.5+0.28

5

13.6_+0.66

5

Valine

7.3+0,63

5

7.2+_0.38

5

Cystelnec

1.0+0.07

2

0.8_+0.35

2

Methlonine

1.5+0.15

4

1.4_+0.17

3

Isoleucine

7.1~I.05

5

7.3+0.49

5

Leucine

15.8_+0.37

5

15.7_+0.98

5

Tyrosine

1.9_+0.36

6

1.7_+0.07

3

Lyslne

8.1+0.38

6

7.6_+0.3l

4

18.3-H).30

3

17.1_~).76

4

Histidine

8.3_+0.13

4

7.3_+0.44

5

Arginine

4.1+0.21

4

4.0_+0.19

5

Tryptophan

1.8+0.20d

3

1.9_+0.07d

3

aAverage value with standard deviation bExtrapolated to zero time CDetermlnedas cysteic acid dDetermined spectrophoton!etrlcelly

Pacific and Atlantic turtle myoglobins, respectively. Values obtained for sperm whale and yellowfin myoglobin controls were in close agreement with those in the literature. The gel filtration, SDS-electrophoresis, and amino acid analyses suggest that both turtle myoglobins are monomers. The values reported in Table 2 for aspartic acid and cysteine and the respective standard deviations suggest a total of amino acid residues per molecule within the general range of 150.

Isoelectric points

8o

~

6°

"

_ jw, 5

10

15

i!

'i

20 25 30 35 40 45 GEL LENGTHtram)

Fig. 4. Spectrophotometric scan of acrylamide gel separation of Pacific green sea turtle myoglobin components showing microheterogeneity and separation of oxy and met forms. Partial scan of sperm whale myoglobin gel shown for comparison. ( ) Pacific turtle Mb; (- . . . . ) sperm whale Mb; A, metMb forms; B, oxyMb forms; O, pH gradient.

M icroheterogeneity As many as 9 minor bands of myoglobin could be seen upon first staining the electrofocusing gels, 8 on the positive side of oxymyoglobin and 1 between oxy and metmyoglobin. The initial reaction product of the staining procedure is bluish in color. Minor bands visible at this point become very diffuse as the staining procedure proceeds to the final reaction product, which is brown. Many of these minor bands were not discernable on the chromatograms obtained from scanning the gels at 409 nm. Attempts to find a more propitious wavelength at which to scan proved unsuccessful. As a result, 6 minor bands were recorded for Pacific turtle myoglobin after Sephadex G-75 purification (Fig. 4) and 3 minor bands after Sephadex CM-50 purification. The samples of Atlantic turtle myoglobin, which were all purified by gel filtration plus ion exchange, exhibited 5 minor bands with the same pattern exhibited by the Pacific turtle myoglobin.

Oxygen equilibrium

Using an acrylamide gel matrix for electrofocusing proved to be a rapid and reproducible method of observing heterogeneity and estimating pI values. Quinn (1973) verified the capacity of acrylamide gel systems to reduce ferrimyoglobin; such reduction was observed with myoglobins used in this study. A major ferromyoglobin band could not be removed even in some samples treated with potassium ferricyanide. Both turtle myoglobins exhibited essentially identical pI values as shown in Table 3. Table 3. Isoelectric points of turtle, whale and tuna myoglobins Myoglobin Source

10.0

Detm.

21.7t0.50

Phenylalanine

257

Isoelectric Pointa Metmyoglobln

Oxymyoglobin 7.40_+0.02

No. of Detm.

Sperm whale

7.93+__0.04

Yellowfln tuna

8.Z5

3

Pacific turtle

7.23+0.04

6.80_+0.06

5

Atlantic turtle

7.23L~0.06

6.83~.02

3

l

aAs determined by electrofocusing in acrylamlde gels

Results of the oxygen equilibrium measurements, when plotted in the conventional fashion of per cent MbO2 vs pO2, showed a hyperbolic curve typical of myoglobins in general. The value of Pl/2 (i.e. oxygen pressure required for 50% saturation of myoglobin) calculated from such plots was 1.30 mm mercury. For control purposes a Pl/2 value of 0.40 was determined for sperm whale myoglobin, compared to a literature value of 0.51 (Antonini & Brunori, 1971); the reference just cited summarizes Pl/2 values for myoglobulins from a variety or sources that range from 0.46 to 1.3. Thus the fact that sea turtles are capable of deep prolonged diving apparently cannot be related in any way to a comparatively high myoglobin oxygen affinity which, presumably, would represent a biological advantage in terms of a more efficient oxygen transport mechanism. Anamolous values were obtained if samples were maintained at a very low oxygen pressure for extended periods, due to increased autoxidation under these conditions. Determination of the 580 nm/542 nm absorbance ratio after complete oxygenation was

258

JOHN D. WILLIAMS, JR. AND W. DUANE BROWN

Table 4. Amino acid composition of myoglobins

Lys

15

15

19

17

18

19

His

5

6

8

7

8

12

9

Acknowledqements--The authors thank Dr. Robert Rice for many helpful suggestions: Mr. Dan Watts for technical assistance: and Mr. Paul Hoekenga and Mr. Roy O. WiIliams for supplying the turtles used in this study. This work was supported in part by the NOAA Office of Sea Grant, Department of Commerce, under Grant No. 04-3-158-22. The U.S. Government is authorized to produce and distribute reprints for governmental purpose notwithstanding any copyright notation that may appear here-

Arg

6

2

4

4

4

4

2

Oil.

Asp

18

13

14

]3

13

8

Thr

12

7

6

8

8

5

4

Ser

4

4

7

9

7

6

7

Glu

14

22

21

22

19

21

Pro

8

7

4

6

5

4

5

G1y

8

14

9

lO

8

II

15

Ala

9

22

15

14

14

17

12

Cys

l

1

0

1

1

0

1

Val

12

8

6

7

7

8

7

Met

3

3

2

2

2

2

3 2

#mlino

Salmon Yellowfinb

Loggerheada

Acid

Sharka

Sea Turtle

Tuna

II

Green

Spermc

Sea Turtle Pac.

Atl.

Human d

Whale

20

II

Tyr

2

1

2

2

2

3

Phe

7

6

8

8

8

6

7

Trp

2

1

2

2

2

2

2

lieu

12

tO

6

7

7

9

8

Leu

16

18

17

16

16

18

17

aTomita and rsuchiya (1971) bFosmi re 0970) CEdmundson (1965) dHerrera and Lehman (I971b)

found valuable in evaluating the quality of data from each experiment attempt. Comparative amino acid composition The sea turtle myoglobins amino acid profiles resemble much more closely those of mammals than those of tuna or shark (Table 4). Preliminary results suggest a blocked N-terminus, as reported for yellowfin tuna myoglobin (Amano et al., 1968).

SUMMARY Myoglobins from Atlantic and Pacific green sea turtles have been purified and characterized. Spectral properties are similar to those of other myoglobins and infrared" spectra indicate that the heine group of turtle myoglobin is protoheme IX. The molecular weight of Pacific sea turtle myoglobin appears to be about 18,000. The two turtle myoglobins have very similar amino acid compositions, and both contain one cysteine residue per molecule in contrast to most other myoglobins. The amino acid profiles of these proteins resemble much more closely those of mammals than those of tuna or shark. Micro,heterogeneity of myoglobins from both turtles could readily be demonstrated by means of electrofocusing gels. Oxygen equilibria studies gave results typical of other myoglobins and do not support any presumption that the capability of these animals for deep prolonged diving can be related to a particularly high myoglobin oxygen affinity.

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