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The hemoglobins of Myxine glutinosa L.—II. amino acid analyses, end group determinations and further investigations
Comp. Biochem. Physiol., 1972, Vol. 42B,pp. 611 to 617. Pergamon Press. Printed in Great Britain
T H E H E M O G L O B I N S OF M Y X I N E GLUTINOSA L . - - I I . A M I N O ACID ANALYSES, END GROUP D E T E R M I N A T I O N S AND F U R T H E R I N V E S T I G A T I O N S SVEN PAL/~US* and G I S E L A L I L J E Q V I S T * Biochemical Department, Nobel Medical Institute, Caroline Institute, S-104 01 Stockholm 60, Sweden (Received 21 December 1971)
Abstract--1. The amino acid composition of the three main hemoglobins, Hb I, Hb II and Hb III, of the hagfish (Myxine glutinosa L.) has been determined. The composition differs so much from that of the lamprey (Lampetra fluviatilis) hemoglobin that a diphyletic origin of the two species seems likely. 2. The N-terminal residue of Hb III (hagfish) is proline as is also that of lamprey hemoglobin. The N-terminal group of Hb I and II seems to be blocked. 3. Because the three main hagfish hemoglobins have different molecular weights the amino acid composition is not sufficient for a discussion of their phylogenetie interrelationship but consideration of the amino acid sequences would be necessary. INTRODUCTION IN A PREVIOUS paper the isolation and crystallization of some of the hagtish hemoglobins have been described (Pal6us et aL, 1971). The three main components were named Hb I, Hb II and Hb III. In this paper the amino acid analyses and end group determinations of these hemoglobins will be presented and their molecular weights discussed. RESULTS
A m i n o acid analyses
Hemoglobins I, II and I I I from the preparations described earlier (Pal6us et al., 1971) were analyzed for amino acids. In the case of Hb I and III crystalline material was used. The hemoglobin fraction was dialyzed againt 0.002 M ammonium hydroxide, evaporated to dryness and then hydrolyzed at 105°C for 20 (in a few cases 23) hr or 70 (or 72) hr with 5.7 N HCI in an evacuated ampoule. After hydrolysis the ampoules were opened and the contents transferred to the flask of a rotatory evaporator, where the hydrochloric acid was effectively removed. The residue was dissolved in 25 ml of a citrate buffer of pH 2.2 used in the chromatographic procedure (Spackman et al., 1958). The curves obtained from the chromatographic procedure were evaluated as described by these authors, but the *Present address: Blodverksamheten, Fack, S-100 64 Stockholm 38, Sweden. 611
Asp Thr Set Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg
0.610 0"238 0"579 1"141 0"330 0"188 0"484 0 0"403 0"095 0"415 0"653 0.144 0-427 0-718 0-146 0.203
70 hr
0-489 0"293 0"436 0"608 0"310 0"194 0"521 0 0"250 0"198 0"239 0"524 0-137 0.327 0.653 0.146 0.074
20 hr 0"489 0"293 0"435 0"607 0"310 0"194 0"519 0 0"250 0"200 0"239 0"525 0.137 0.327 0"654 0"146 0.074
70 hr
Hb I I I
0-742 0"375 0"532 0"924 0"149 0"398 0"741 0 0"458 0"252 0"435 0"504 0.141 0-615 0-678 0-297 0.319
31 hr
Hb II
0"692 0-384 0"630 1"011 0"439 0"290 0"720 0 0"349 0"231 0"360 0"676 0.198 0.448 0"861 0"194 0.112
24 hr 0"692 0"387 0"631 1"016 0"~ 0"290 0"719 0 0"351 0"231 0"364 0"680 0.191 0.446 0.865 0.194 0.112
56 hr
Hb III
0.0655 0"0241 0"0587 0"1032 0"0365 0"0263 0"0489 0 0"0421 0"0103 0"0454 0"0680 0-0186 0.0462 0.0644 0.0142 0"0202
23 hr 0.0656 0"0241 0"0531 0"1031 0"0365 0"0264 0"0484 0 0"0409 0 0"0454 0"0678 0.0134 0.0462 0-0643 0.0143 0-0203
72 hr
Hb I
0.0691 0"0350 0"0470 0"0861 0"0159 0"0377 0"0762 0 0"0467 0"0187 0"0478 0"0560 0.0156 0.0402 0.0697 0.0308 0.0308
0"0689 0"0351 0"0470 0"0861 0"0160 0'0376 0"0767 0 0'0464 0"0188 0"0480 0"0558 0.0156 0'0402 0"0697 0"0307 0"0309
72 hr
H b II
Preparation 3
23 hr
C O M P O S I T I O N OF HAGFISH H E M O G L O B I N S *
Preparation 2
*Values expressed in/zmoles/ml sample solution. tHydrolysis time in hours.
0-610 0-238 0"579 1"140 0"330 0"188 0"484 0 0"403 0"099 0"415 0"653 0.145 0.427 0-718 0"146 0.203
20 hr
Hb I
Preparation 1
TABLE 1--AMINO ACID
0-1819 0"1017 0'1510 0"2324 0"1042 0"0714 0'1883 0 0"0816 0"0487 0"0828 0"1828 0.0501 0.1144 0.2210 0.0469 0.0228
23 hr
0.1854 0"1023 0"1658 0"2368 0"1055 0"0729 0"1918 0 0"0866 0"0492 0"0974 0"1832 0.0520 0.1155 0.2253 0.0488 0.0256
72 hr
Hb I I I
>
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Io
THE HEMOGLOBINS OF M Y X I N E
613
GLUT1NOSA L.--II
primary data were treated in a computer by a special program worked out by Dr. B. Lindqvist (Mj61kcentralen, Stockholm). This treatment gives the amounts of /zmoles of each amino acid (Table 1). F r o m these values the most probable amino acid residue numbers were calculated by the program in a least-squares mode, using conventional calculation techniques. D u r i n g these least-squares mode calculations various interpretations of the analyses can be made, each representing a near whole-number multiple of the first composition obtained. I n Table 2 one TABLE 2 - - A M I N O
ACID COMPOSITION OF HAGFISH HEMOGLOBINS COMPARED W I T H THAT OF
LAMPREY HEMOGLOBIN~ TOTAL NUMBER OF AMINO ACID RESIDUES GIVING BEST AGREEMENT W I T H THE MOLECULAR WEIGHTS AS DETERMINED BY ULTRACENTRIFUGATION*
Preparation 1
Preparation 2
Preparation 3
Hemoglobin
Hemoglobin
Hemoglobin
I
III
II
pH
Hemoglobin of L. fluviatilis
III
I
II pH
III
pH
5"0
8"0
6"7
8"0
4'9
6"8
8"5
26 10 24 48 14 8 20 0 16 4 18 28 6 18 30 6 8
17 11 15 21 11 7 18 0 9 7 9 18 5 12 23 5 3
20 10 14 24 4 10 20 0 12 6 12 14 4 16 18 8 8
17 10 16 25 11 7 18 0 8 6 8 17 5 11 22 5 2
24 9 21 37 13 10 18 0 15 4 16 24 7 17 23 5 7
18 9 12 23 4 10 20 0 12 5 13 15 4 11 18 8 8
18 10 16 22 10 7 18 0 8 5 9 17 5 11 21 5 2
14 7 15 10 6 6 20 1 12 6 8 8 4 8 13 2 4 2
Total numbers 284 of residues
191
200
188
250
190
184
146
Asp Thr Ser Glu Pro Gly Ala Cyst Val Met Ile Leu Tyr Phe Lys His Arg Trp
*(Quast et al., 1969). The methods of preparation indicated by Arabic numerals have been described previously (Pal~us et al., 1971). tThese analyses were made on preparations without pretreatment with performic acid. ++Braunitzer & Fujiki (1969).
614
SWN PAL~.USAND GISELA LILJEQVIST
such solution is presented; here the number of amino acid residues is adapted to the molecular weights obtained by an ultracentrifugal study. End group determinations The N-terminal group was determined by the dansyl-Edman method (Gray & Hartley, 1967; JOrnvaU & Harris, 1970). After reaction of the protein with 1dimethylaminonaphthalene-5-sulphonyl chloride the identification of the dansyl amino acids was made by thin-layer chromatography. Hb III has proline as the N-terminal group, whereas the N-terminal residues of Hb I and Hb II seem to be blocked. Preliminary experiments using the carboxypeptidase method (Guidotti et al., 1962) showed serine to be the C-terminal in Hb I and lysine in Hb II and Hb III. Further analyses Iron determinations of Hb I and Hb II (from Preparation 3) showed 0.24 and 0.25 per cent, and determinations on Hb III (from Preparations 2 and 3) 0.26 and 0.31 per cent, respectively. Tryptophan, determined by the method of Bates (1937), was not detected in Hb I, II or III. A Beckman Microzone Electrophoresis, using veronal buffer of pH 8.6 and Na-phosphate buffer of pH 6.0, revealed two components in Hb I (Preparation 2), three in Hb II and one component in Hb III. In the case of Preparation 3, Hb I and Hb III appeared as single components while the interpretation of the heterogeneity of Hb II was ambiguous. Electrophoresis of Hb II in a Tiselius apparatus showed this fraction to be non-homogeneous in agreement with previous work (Pal6us et al., 1971) where we observed three subfractions of Hb II. This explains why no crystals were obtained from this fraction. Absorption spectra in the visible and u.v. regions were made on the oxyform of Hb I from Preparation 3 (Fig. 1). The pyridine hemochromogen showed its maximum at 556 nm.
DISCUSSION As early as 1966 the polymorphism of hagfish hemoglobin was described (Pal6us & Vesterberg, 1966). At about the same time Braunitzer's group published similar observations on the hemoglobin of insect larvae from Chironomus (Braun et al., 1968). In an earlier paper (Pal6us et al., 1971) we have described the separation of more than ten fractions of hagfish hemoglobin, two of which, Hb I and Hb III, have been crystallized. An additional main fraction, Hb II, has been isolated but all attempts to crystallize it have hitherto failed. Electrophoretic investigations and the present amino acid analyses both indicate that this fraction is non-homogeneous. By ultracentrifugation (Quast et al., 1969) we have shown that the average molecular weights of the three isolated hemoglobins are 28,000, 21,000 and 20,500 respectively. The minimal molecular weights according to the iron determinations are 23,300, 22,300 and 19,400, respectively, also showing the molecular weights to decline from Hb I, Hb II to Hb Ill. The computer treatment
THE HEMOGLOBINS OF M Y X I N E
GLUT1NOS.~ L.--II
615
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f
O
O
o
O
¢n
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616
SVEN PALI~USAND GISELA LILJEQVIST
of the amino acid analyses in the case of Preparation 3 gave molecular weight values of 20,473 for Hb III when a 23-hr hydrolysate was used, and 20,356 when a 72-hr hydrolysate was used. For Hb I, molecular weights of 27,913 obtained from a 23-hr hydrolysate and 27,778 from a 72-hr hydrolysate represent interpretations of the amino acid analyses with relevant small statistical errors. For comparison it should be mentioned that the main fraction of the lamprey hemoglobin has a molecular weight of around 19,000 (Rudloff et al., 1966). Usually the smaller respiratory pigments with molecular weights of 70,000 or less are intracellular, as in the case of the Cyclostomes, while respiratory pigments with molecular weights larger than 70,000 are extracellular and show a large polymorphism. We consider the above findings to be compatible with the interpretation that at least four independent loci of genes exist for the monomeric hemoglobin in the hagfish (Ohno & Morrison, 1966). The interpretation of the described polymorphism is difficult, but we consider it as a primitive feature and as a phase of molecular evolution (Kimura & Ohta, 1971), which might be associated with the transformation from marine to terrestrial life and which may also reflect Nature's great experiment with gene duplication (Ohno, 1970) and the special conditions for loading of oxygen in this primitive animal. Refined analytical procedures have indicated also the existence of more than one type of specific chain among the mammalian hemoglobins (Huisman, 1969). It may be mentioned here that the hemoglobin of the cyclostome, Lampetra fluviatilis (Fujiki & Braunitzer, 1970), consists of a single monomeric chain, while that of Petromyzon marinus (Rumen & Love, 1963) consists of six polypeptide chains. This may be explained by different environmental conditions for variants of the same species. Whether the polymorphism of salmon hemoglobin (Wilkins, 1970) can be explained by the special living conditions of this species is still not clear. Acknowledgements--The authors wish to express their sincere gratitude to Professor H. Theorell for criticism in connection with the work. It is also a pleasure to thank Mrs. B. Lundqvist for skilful technical assistance. This work was supported by grants from the Swedish National Science Research Council.
REFERENCES BATESR. W. (1937) A rapid method for quantitative determination of tryptophane. 9%biol. Chem. 119, VII. BRAUNV., CRmHTONR. R. & BRAUNITZERG. (1968) ~ber monomere und dimere Insektenh~noglobine (Chironomus thummi). Z. physiol. Chem. 349, 197-210. BRAUNtTZ~ G. & FUJtKI H. (1969) Zur evolution der Vertebraten die konstitution und terti~lrstukter des hiimoglobins des flussneunauges. Naturwiss, 56, 322-323. FUJIKI H. & Bm~UNXTZm~G. (1970) The primary structure of lamprey haemoglobin and its contribution to the evolutionary aspects of haemoglobin molecules. Folia Bioch. Biol. Graeca 7, 68-73. GRAYW. R. & HARTLEYB. S. (1963) The structure of a chymotryptic peptide from Pseudomonas cytochrome c-551. A fluorescent end-group reagent for proteins and peptides. Biochem..7. 89, 379-380 and 59P.
THE HEMOGLOBINS OF M Y X I N E G L U T I N O S A L . - - I I
617
GUIDOTTIG., HILL R. & KONINGSBERGW. (1962) The structure of human hemoglobin--II. The separation and amino acid composition of the tryptic peptides from the ct- and fl-chains. ~t. biol. Chem. 237, 2184-2195. HUISMANT. H. J. (1969) Multiple ~t and fl chain structural genes as a basis for hemoglobin heterogeneity of the adult goat. In Protides of Biological Fluids (Edited by PEETERSH.) Vol. 17, pp. 241-248. J~RNVALL H. ~ HARRISJ. I. (1970) Horse liver alcohol dehydrogenase. On the primary structure of the ethanol-active isoenzyme. Eur.j7. Biochem. 13, 565-576. KIMURA M. & OHTA T. (1971) Protein polymorphism as a phase of molecular evolution. Nature, Lond. 229, 467-469. OHNO S. (1970) Evolution by Gene Duplication. Springer-Verlag, Berlin, 124-132. OHNO S. & MORmSON M. (1966) Multiple gene loci for the monomeric hemoglobin of the hagfish (Eptatretus stoutii). Science 154, 1034-1035. PALI~US S. 86 VESTERBERGO. (1966) The multiplicity of Myxine glutinosa hemoglobin, revealed by a new separation method. International Symposium on Comparative Hemoglobin Structure, Thessaloniki, 11-13 April 1966. (Edited by POLYCHRONAKOSD. J.), pp. 149-150. Triantafylou, Thessaloniki, Greece. PALI~US S., VESTERBERGO. • LILJEQVIST G. (1971) The hemoglobins of Myxine glutinosa L.--I. Preparation and crystallization. Comp. Biochem. Physiol. 39B, 551-557. QUAST R., PAL~USS., BLOOMG. & OSTLUNDE. (1969) A study on the molecular weight of hemoglobin from Myxine glutinosa L. ,4cta Chem. Scan& 23, 3595-3596. RUDLOFF V., ZELENIK M. & BaAUNITZER G. (1966) Zur Phylogenie des H~imoglobinmolekiils. Untersuchungen am H~imoglobin des Flussneunauges (Lampetra fluviatilis). Z. physiol. Chem. 344, 284-288. RUMENN. & L o w W. (1963) The six hemoglobins of the sea lamprey (Petromyzon marinus). .4rehs. Biochem. Biophys. 103, 24-35. SFACKMAND. H., STEINW. H. & M o o ~ S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. WILKINSN. P. (1970) The sub-unit composition of the haemoglobins of the Atlantic salmon (Salmo salar L.). Biochim. biophys. Acta 214, 52-63.
Key Word Index--Hemoglobin of Myxine; amino acid analyses; end group determinations; Myxine glutinosa.
T H E H E M O G L O B I N S OF M Y X I N E GLUTINOSA L . - - I I . A M I N O ACID ANALYSES, END GROUP D E T E R M I N A T I O N S AND F U R T H E R I N V E S T I G A T I O N S SVEN PAL/~US* and G I S E L A L I L J E Q V I S T * Biochemical Department, Nobel Medical Institute, Caroline Institute, S-104 01 Stockholm 60, Sweden (Received 21 December 1971)
Abstract--1. The amino acid composition of the three main hemoglobins, Hb I, Hb II and Hb III, of the hagfish (Myxine glutinosa L.) has been determined. The composition differs so much from that of the lamprey (Lampetra fluviatilis) hemoglobin that a diphyletic origin of the two species seems likely. 2. The N-terminal residue of Hb III (hagfish) is proline as is also that of lamprey hemoglobin. The N-terminal group of Hb I and II seems to be blocked. 3. Because the three main hagfish hemoglobins have different molecular weights the amino acid composition is not sufficient for a discussion of their phylogenetie interrelationship but consideration of the amino acid sequences would be necessary. INTRODUCTION IN A PREVIOUS paper the isolation and crystallization of some of the hagtish hemoglobins have been described (Pal6us et aL, 1971). The three main components were named Hb I, Hb II and Hb III. In this paper the amino acid analyses and end group determinations of these hemoglobins will be presented and their molecular weights discussed. RESULTS
A m i n o acid analyses
Hemoglobins I, II and I I I from the preparations described earlier (Pal6us et al., 1971) were analyzed for amino acids. In the case of Hb I and III crystalline material was used. The hemoglobin fraction was dialyzed againt 0.002 M ammonium hydroxide, evaporated to dryness and then hydrolyzed at 105°C for 20 (in a few cases 23) hr or 70 (or 72) hr with 5.7 N HCI in an evacuated ampoule. After hydrolysis the ampoules were opened and the contents transferred to the flask of a rotatory evaporator, where the hydrochloric acid was effectively removed. The residue was dissolved in 25 ml of a citrate buffer of pH 2.2 used in the chromatographic procedure (Spackman et al., 1958). The curves obtained from the chromatographic procedure were evaluated as described by these authors, but the *Present address: Blodverksamheten, Fack, S-100 64 Stockholm 38, Sweden. 611
Asp Thr Set Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg
0.610 0"238 0"579 1"141 0"330 0"188 0"484 0 0"403 0"095 0"415 0"653 0.144 0-427 0-718 0-146 0.203
70 hr
0-489 0"293 0"436 0"608 0"310 0"194 0"521 0 0"250 0"198 0"239 0"524 0-137 0.327 0.653 0.146 0.074
20 hr 0"489 0"293 0"435 0"607 0"310 0"194 0"519 0 0"250 0"200 0"239 0"525 0.137 0.327 0"654 0"146 0.074
70 hr
Hb I I I
0-742 0"375 0"532 0"924 0"149 0"398 0"741 0 0"458 0"252 0"435 0"504 0.141 0-615 0-678 0-297 0.319
31 hr
Hb II
0"692 0-384 0"630 1"011 0"439 0"290 0"720 0 0"349 0"231 0"360 0"676 0.198 0.448 0"861 0"194 0.112
24 hr 0"692 0"387 0"631 1"016 0"~ 0"290 0"719 0 0"351 0"231 0"364 0"680 0.191 0.446 0.865 0.194 0.112
56 hr
Hb III
0.0655 0"0241 0"0587 0"1032 0"0365 0"0263 0"0489 0 0"0421 0"0103 0"0454 0"0680 0-0186 0.0462 0.0644 0.0142 0"0202
23 hr 0.0656 0"0241 0"0531 0"1031 0"0365 0"0264 0"0484 0 0"0409 0 0"0454 0"0678 0.0134 0.0462 0-0643 0.0143 0-0203
72 hr
Hb I
0.0691 0"0350 0"0470 0"0861 0"0159 0"0377 0"0762 0 0"0467 0"0187 0"0478 0"0560 0.0156 0.0402 0.0697 0.0308 0.0308
0"0689 0"0351 0"0470 0"0861 0"0160 0'0376 0"0767 0 0'0464 0"0188 0"0480 0"0558 0.0156 0'0402 0"0697 0"0307 0"0309
72 hr
H b II
Preparation 3
23 hr
C O M P O S I T I O N OF HAGFISH H E M O G L O B I N S *
Preparation 2
*Values expressed in/zmoles/ml sample solution. tHydrolysis time in hours.
0-610 0-238 0"579 1"140 0"330 0"188 0"484 0 0"403 0"099 0"415 0"653 0.145 0.427 0-718 0"146 0.203
20 hr
Hb I
Preparation 1
TABLE 1--AMINO ACID
0-1819 0"1017 0'1510 0"2324 0"1042 0"0714 0'1883 0 0"0816 0"0487 0"0828 0"1828 0.0501 0.1144 0.2210 0.0469 0.0228
23 hr
0.1854 0"1023 0"1658 0"2368 0"1055 0"0729 0"1918 0 0"0866 0"0492 0"0974 0"1832 0.0520 0.1155 0.2253 0.0488 0.0256
72 hr
Hb I I I
>
©
> ~,
Io
THE HEMOGLOBINS OF M Y X I N E
613
GLUT1NOSA L.--II
primary data were treated in a computer by a special program worked out by Dr. B. Lindqvist (Mj61kcentralen, Stockholm). This treatment gives the amounts of /zmoles of each amino acid (Table 1). F r o m these values the most probable amino acid residue numbers were calculated by the program in a least-squares mode, using conventional calculation techniques. D u r i n g these least-squares mode calculations various interpretations of the analyses can be made, each representing a near whole-number multiple of the first composition obtained. I n Table 2 one TABLE 2 - - A M I N O
ACID COMPOSITION OF HAGFISH HEMOGLOBINS COMPARED W I T H THAT OF
LAMPREY HEMOGLOBIN~ TOTAL NUMBER OF AMINO ACID RESIDUES GIVING BEST AGREEMENT W I T H THE MOLECULAR WEIGHTS AS DETERMINED BY ULTRACENTRIFUGATION*
Preparation 1
Preparation 2
Preparation 3
Hemoglobin
Hemoglobin
Hemoglobin
I
III
II
pH
Hemoglobin of L. fluviatilis
III
I
II pH
III
pH
5"0
8"0
6"7
8"0
4'9
6"8
8"5
26 10 24 48 14 8 20 0 16 4 18 28 6 18 30 6 8
17 11 15 21 11 7 18 0 9 7 9 18 5 12 23 5 3
20 10 14 24 4 10 20 0 12 6 12 14 4 16 18 8 8
17 10 16 25 11 7 18 0 8 6 8 17 5 11 22 5 2
24 9 21 37 13 10 18 0 15 4 16 24 7 17 23 5 7
18 9 12 23 4 10 20 0 12 5 13 15 4 11 18 8 8
18 10 16 22 10 7 18 0 8 5 9 17 5 11 21 5 2
14 7 15 10 6 6 20 1 12 6 8 8 4 8 13 2 4 2
Total numbers 284 of residues
191
200
188
250
190
184
146
Asp Thr Ser Glu Pro Gly Ala Cyst Val Met Ile Leu Tyr Phe Lys His Arg Trp
*(Quast et al., 1969). The methods of preparation indicated by Arabic numerals have been described previously (Pal~us et al., 1971). tThese analyses were made on preparations without pretreatment with performic acid. ++Braunitzer & Fujiki (1969).
614
SWN PAL~.USAND GISELA LILJEQVIST
such solution is presented; here the number of amino acid residues is adapted to the molecular weights obtained by an ultracentrifugal study. End group determinations The N-terminal group was determined by the dansyl-Edman method (Gray & Hartley, 1967; JOrnvaU & Harris, 1970). After reaction of the protein with 1dimethylaminonaphthalene-5-sulphonyl chloride the identification of the dansyl amino acids was made by thin-layer chromatography. Hb III has proline as the N-terminal group, whereas the N-terminal residues of Hb I and Hb II seem to be blocked. Preliminary experiments using the carboxypeptidase method (Guidotti et al., 1962) showed serine to be the C-terminal in Hb I and lysine in Hb II and Hb III. Further analyses Iron determinations of Hb I and Hb II (from Preparation 3) showed 0.24 and 0.25 per cent, and determinations on Hb III (from Preparations 2 and 3) 0.26 and 0.31 per cent, respectively. Tryptophan, determined by the method of Bates (1937), was not detected in Hb I, II or III. A Beckman Microzone Electrophoresis, using veronal buffer of pH 8.6 and Na-phosphate buffer of pH 6.0, revealed two components in Hb I (Preparation 2), three in Hb II and one component in Hb III. In the case of Preparation 3, Hb I and Hb III appeared as single components while the interpretation of the heterogeneity of Hb II was ambiguous. Electrophoresis of Hb II in a Tiselius apparatus showed this fraction to be non-homogeneous in agreement with previous work (Pal6us et al., 1971) where we observed three subfractions of Hb II. This explains why no crystals were obtained from this fraction. Absorption spectra in the visible and u.v. regions were made on the oxyform of Hb I from Preparation 3 (Fig. 1). The pyridine hemochromogen showed its maximum at 556 nm.
DISCUSSION As early as 1966 the polymorphism of hagfish hemoglobin was described (Pal6us & Vesterberg, 1966). At about the same time Braunitzer's group published similar observations on the hemoglobin of insect larvae from Chironomus (Braun et al., 1968). In an earlier paper (Pal6us et al., 1971) we have described the separation of more than ten fractions of hagfish hemoglobin, two of which, Hb I and Hb III, have been crystallized. An additional main fraction, Hb II, has been isolated but all attempts to crystallize it have hitherto failed. Electrophoretic investigations and the present amino acid analyses both indicate that this fraction is non-homogeneous. By ultracentrifugation (Quast et al., 1969) we have shown that the average molecular weights of the three isolated hemoglobins are 28,000, 21,000 and 20,500 respectively. The minimal molecular weights according to the iron determinations are 23,300, 22,300 and 19,400, respectively, also showing the molecular weights to decline from Hb I, Hb II to Hb Ill. The computer treatment
THE HEMOGLOBINS OF M Y X I N E
GLUT1NOS.~ L.--II
615
o ¢-
o
¢d
~o O
..Q
6
f
O
O
o
O
¢n
9
/;~ !suep
]~3!ido
616
SVEN PALI~USAND GISELA LILJEQVIST
of the amino acid analyses in the case of Preparation 3 gave molecular weight values of 20,473 for Hb III when a 23-hr hydrolysate was used, and 20,356 when a 72-hr hydrolysate was used. For Hb I, molecular weights of 27,913 obtained from a 23-hr hydrolysate and 27,778 from a 72-hr hydrolysate represent interpretations of the amino acid analyses with relevant small statistical errors. For comparison it should be mentioned that the main fraction of the lamprey hemoglobin has a molecular weight of around 19,000 (Rudloff et al., 1966). Usually the smaller respiratory pigments with molecular weights of 70,000 or less are intracellular, as in the case of the Cyclostomes, while respiratory pigments with molecular weights larger than 70,000 are extracellular and show a large polymorphism. We consider the above findings to be compatible with the interpretation that at least four independent loci of genes exist for the monomeric hemoglobin in the hagfish (Ohno & Morrison, 1966). The interpretation of the described polymorphism is difficult, but we consider it as a primitive feature and as a phase of molecular evolution (Kimura & Ohta, 1971), which might be associated with the transformation from marine to terrestrial life and which may also reflect Nature's great experiment with gene duplication (Ohno, 1970) and the special conditions for loading of oxygen in this primitive animal. Refined analytical procedures have indicated also the existence of more than one type of specific chain among the mammalian hemoglobins (Huisman, 1969). It may be mentioned here that the hemoglobin of the cyclostome, Lampetra fluviatilis (Fujiki & Braunitzer, 1970), consists of a single monomeric chain, while that of Petromyzon marinus (Rumen & Love, 1963) consists of six polypeptide chains. This may be explained by different environmental conditions for variants of the same species. Whether the polymorphism of salmon hemoglobin (Wilkins, 1970) can be explained by the special living conditions of this species is still not clear. Acknowledgements--The authors wish to express their sincere gratitude to Professor H. Theorell for criticism in connection with the work. It is also a pleasure to thank Mrs. B. Lundqvist for skilful technical assistance. This work was supported by grants from the Swedish National Science Research Council.
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Key Word Index--Hemoglobin of Myxine; amino acid analyses; end group determinations; Myxine glutinosa.