A comparison of molecular weights of transferrins of various vertebrates

Comp. Biochem. Physiol. Vol. 79B, No. 1, pp. 113 117, 1984

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A COMPARISON OF MOLECULAR WEIGHTS OF TRANSFERRINS OF VARIOUS VERTEBRATES PETR BOB.~K, ANTONiN STRATIL and MILOSLAV VALENTA Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, 277 21 Lib6chov, Czechoslovakia

(Recewed 1 February 1984) Abstract--1. SDS-polyacrylamide gel electrophoresis has been used to study molecular weights of transferrins of 27 fish, 6 avian and 14 mammalian species. 2. Mol. wts of fish transferrins ranged from 61,000 to 87,000. 3. The values for avian transferrins were in the range from 70,000 to 72,000. 4. For mammalian transferrins mol. wts were estimated to range from 69,000 to 77,000.

INTRODUCTION

The transferrins, homologous iron-binding proteins of blood serum, have been found in all classes of vertebrates. In spite of intensive studies of numerous aspects of transferrin's properties and functions, a comparison of mol. wts of transferrins from various vertebrates on a large scale has not yet been performed, although some limited results are available (e.g. Palmour and Sutton, 1971; Hudson et al., 1973). In view of our earlier study (Stratil et al., 1983b), in which relatively great differences were found in mol. wts o f transferrins o f cyprinid fishes, we decided to compare mol. wts of transferrins of various species of fish, birds and mammals by using a single m e t h o d - SDS-polyacrylamide gel electrophoresis. The tool. wts of transferrins from particular species have already been determined on many occasions, but the results are difficult to compare as different authors used different methods (gel filtration, SDSpolyacrylamide gel electrophoresis, sedimentation equilibrium ultracentrifugation etc.). F o r example, for human transferrin the values range from 66,000 to 95,000 (for references, see Morgan, 1974), while the value calculated from the primary structure is 79,550 (MacGillivray et al., 1982).

system, denaturation of the samples, fixation, staining and destaining were the same as described before (Stratil et al., 1983b). Pre-electrophoresis (without the samples) lasted 10 min at 200 V; electrophoresis was conducted for 3 hr at 250 V (power supply 2103; LKB). For calibration the Low Molecular Weight (LMW), High Molecular Weight (HMW) Electrophoresis Calibration Kits (Pharmacia Fine Chemicals) and human serum transferrin (mol. wt 80,000) were used, Relationships between the relative mobility of the bands (Rf) of the calibrating proteins and logarithm of their mol. wts were linearized by the logit transformation. Equations of regression lines were then computed by the method of least squares and tested using the correlation coett~cient as a critical value. The equations that did not prove suitable were excluded. Mean values of Rswere used for computing of an equation of the calibration line. For calculation of mol. wts of transferrins studied mean values of their Rf were used.

RESULTS

An example of S D S - P A G E of transferrins of various species is shown in Fig. 1 and the results of mol. wt estimations are presented in Table 1. The resulting separation of transferrin reduced with fl-mercaptoethanol did not differ significantly from that without fl-mercaptoethanol. (Transferrins of A.

MATERIALS AND M~THODS

anguilla, S. erythrophthalmus, A. alburnus, V. vimba and L. Iota were studied without fl-mercaptoethanol

Pure or partially purified transferrins from the vertebrate species given in Table 1 were used. If there were some slight impurities in the preparations these should have not interfered with the electrophoretic mobility of the transferrins. Basically, two isolation procedures were used, either Rivanol-ammonium sulphate precipitation with subsequent chromatography on DEAE-Sephadex A-50, or gel filtration of the serum on Sephadex G-100 followed by chromatography on DEAE-Sephadex A-50. In many cases the transferrins were further purified on SP-Sephadex C-50 (for details see, e.g. Stratil and Spooner, 1971; Stratil et al., 1983b). In some cases genetically defined transferrin phenotypes were used, in others a mixture of transferrin variants was employed. Mol. wts of transferrins were estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (SDS-PAGE), with or without fl-mercaptoethanol, in 10.5% acrylamide gels. The buffer

only.) Transferrins of 27 species from six orders of teleostean fishes, six species from three orders of birds and 14 species from seven orders of mammals were compared. The values of tool. wts range from 61,000 (Carassius auratus gibelio) to 87,000 (Esox lucius). The greatest variation in mol. wts was found in fishes. In fishes of the family Cyprinidae, which were studied most extensively (16 species), great differences in mol. wts were found (61,000--79,000). However, the mol. wts of cyprinid transferrins can be clustered into three groups: 61,000-63,000 (the species of the subfamily Cyprininae, Chondrostoma nasus and Tinca tinca); 67,000-70,000 (the species of the subfamily Leuciscinae); and 73,000-79,000 (the species of the subfamilies Barbinae and Hypophthalmichthyinae). Mol. wts of transferrins of the fishes belonging to

CBP/B 79A

H

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PETR BOB~,K et al. Table I. Molecular weights of transferrins of various vertebrates as estimated by SDS-polyacrylamide gel electrophoresis PISCES Anguilliformes Anguillu anguillu Salmoniformes Esox lucius Thymallus thymallus Salmo gairdneri Salvelinus fontinalis Cypriniformes Cyprinus carpio Carassius carassius Carassius auratus gibelio Barbus barbus Barbus meridionalis Leuciscus cephalus Rutilus rutilus Scardinius eo'throphthalmus Aspius asT~ius Alburnus alburnus Vimba vimba Abramis brama Blicca bjoerkna Chondrostoma nasus Ctenophao,ngodon idella Tinca tinca 14vpophthalmichthys molitrix Aristichthys nobilis Siluriformes Silurus glanis Gadiformes Lota lota Perciformes Perca [tuviatilis Stizostedion t,olgense

AVES Anseriformes Anser anser Cairina moschata Anas plat.vrhynchos Galliformes Gallus gallus Meleagris gallopavo Columbiformes Columba livia MAMMALIA Insectivora Erinaceus europaeus Carnivora Canis jamiliaris Felis catus Lagomorpha Oryctolagus cuniculus Rodentia Mesocricetus auratus Rattus norvegicus" Mus musculus Cavia porcellus Perissodactyla Equus caballus Artiodactyla Sus scr~lh Capra hircus Ovis aries Bos taurus Primates Homo sapiens

75,000 87,000 69,000 65.000 84.000 62.000 63.000 61,000 73.000 76,000 68,000 67~000 67.000 69,000 69.000 69,000 66.000 70.000 63,000 73.000 63,000 79,000 76,000 77,000 80,000 74,000 79,000

70.000 71~000 70,000 72,000 7(k000 70,000 69~0(10 70,000 72,000 75,000 75,00(I 74,000 73,000 75.00(1 71,000: 75,000 72,000 71,00(I 70,000 70,000; 74,000 77,000 j

~For the sake of comparison, the mol. w'~ of human transferrin was also calculated from the equation of the calibration line.

b ÷

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O i

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a O

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O

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Fig. 1. (a,b) SDS-polyacrylamide gel electrophoresis of various transferrins. From left to right: (a~ Carassius carassius ; Cyprinus carpio ; E s o x lucius; Silurus glanis ; Chondrostoma nasus ; Ctenopharyngodon idella; L o t a lota; Leuciscus cephalus; Perca fluviatilis; huma n serum transferrin: L M W calibration kit; H M W calibration kit. (b) Bos taurus; L M W calibration kit; Sus scrofa; Oryctolagus cuniculus; Rattus norvegicus ; Felis catus ; Erinaceus europaeus ; huma n serum transferrin; Columba livia ; Anser anser ; Gallus gallus; H M W calibration kit. From H M W calibration kit the following proteins were used: L D H subunit (36,000), catalase subunit (60,000) and bovine serum albumin (67,000).

Molecular weights of various transferrins

Table 2. A survey of published values of molecular weights of transferrins from various species CYCLOSTOMATA Eptatretus stoutii 41,500--45,500 Palmour and Sutton (1971) 76,000 Aisen et al. (1972) Petromyzon marinus 73,200-78,000 Webster and Pollara (1969) PISCES Scyllium stellare lctalurus melas Salvelinus fontinalis Cyprinus carpio

77,500 85,000-95,000 25,500-140,000 58,000-70,000

Tinca tinca

62,000-81,000

Ctenopharyngodon idella Chondrostoma nasus Hypophthalmichthys molitrix Aristichthys nobilis Barbus barbus Barbus meridionalis

72,000; 75,200 62,000 78,000; 78,800 74,000; 78,800 74,000 76,000

Got et al. (1967) Marneux (1972) Hershberger (1970) Silberzahn et al. (1967); Stratil et al. (1983b); Valenta et al. (1976) Stratil et al. (1983b); Eijk et al. (1972) Stratil et al. (1983b) Stratil et al. (1983b) Stratil et al. (1983b) Stratil et al. (1983b) Stratil et aL (1983a) Stratil et al. (1983a)

78,000 72,000-81,000

Foucrier et al. (1976) Palmour and Sutton (1971)

85,000-96,000 68,000-77,800

Palmour and Sutton (1971) Makey and Seal (1970)

60,000

Stratil and Spooner (1971) Bezkorovainy et al. (1968); Bezkorovaiuy and Grohlich 0974); Fuller and Briggs 0956); Stratil and Spoouer (1971); Greene and Feeney (1968); Williams et al. (1982) Frelinger (1973)

AMPHIBIA Pleurodeles waltlii Rana catesbiana

REPTILIA Pseudemys scripta Eunectes murinus

AVES Arias platyrhynchos i Gallus gallus t

Columba livia

74,000-84,000

77,7702 80,000

MAMMALIA Homo sapiens

66,000-95,000

Papio leucophaeus Macaeus rhesus Rattus norvegicus

79,5502 77,000 68,500; 70,800 66,100-83,000

Mus musculus Equus caballus

66,700 70,000-80,500

Oryctolagus cuniculus

70,000-82,000

Sus scrofa

74,00(O88,000

Ovis aries

77,000; 77,500

Bos taurus

67,000-77,500

~Ovotransferrin. 2The values calculated from the primary structures.

for references see, e.g. Morgan (1974) MacGillivray et aL (1982) Bezkorovaiuy and Grohlich (1974) Charlwood (1963) Charlwood (1963); Buglanov et al. (1980); Sehreiber et aL (1979); Eijk et aL (1972) Watkins et aL (1966) Hudson et al. 0973); Stratil et aL (1984) Baker et aL (1968); Hudson et al. (1973); Palmour and Sutton (1971); Greene and Feeney (1968); Eijk et aL (1972) Stratil and Spoouer (1971); Hudson et aL (1973); Valenta et al. 0976); Leibman and Aisen (1967); Laurell and Ingelman (1947) Gu6rin et al. (1976); Spoouer et al. (1975) Efremov et aL 0971); Brock et aL (1976; 1978); Stratil and Spoouer (1971); Hatton et al. (1977); Bezkorovainy and Grohlich 0974); Spooner et al. 0975); Hudson et al. (1973); Richardson et al. (1973); Tsuji et al. (1981)

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PETR BOBJ,K el al.

other orders differ from one a n o t h e r , so that no relationship can be seen, a n d moreover, the n u m b e r of species studied was too low. Mol. wts o f avian transferrins (orders Anseriformes, Galliformes and C o l u m b i f o r m e s ) were very similar to one a n o t h e r (range from 70,000 to 72,000). Mol. wts of m a m m a l i a n transferrins were in a more n a r r o w range t h a n the fish transferrins. M o s t of the values ranged from 70,000 to 77,000. A n exception was the transferrin o f Erinaceus europaeus (69,0003. In bovine (Bos taurus) transferrin two zones were observed c o r r e s p o n d i n g to mol. wts of 70,000 a n d 74,000, respectively. Two zones (tool. wts 71,000 and 75,000, respectively) were also seen in horse (Equus caballus ) transferrin. DISCUSSION The technique o f S D S - P A G E , which was used for estimation of mol. wts of transferrins, provides relative values of mol, wts, which are the result of the relationship between the mol. wt o f S D S - d e n a t u r a t e d proteins a n d electrophoretic migration. This m e t h o d is especially useful for a parallel c o m p a r i s o n of a large n u m b e r o f h o m o l o g o u s proteins. It is relatively simple, reproducible, reliable a n d the values o b t a i n e d c o m p a r e well. It is clear t h a t the mol. wts o b t a i n e d by S D S - P A G E may differ from the values o b t a i n e d by o t h e r m e t h o d s (sedimentation equilibrium ultracentrifugation, gel filtration etc.). This explains the differences between our results a n d those of o t h e r a u t h o r s ( c o m p a r e Tables 1 a n d 2). Exceptional two-zone p a t t e r n s were observed in b o v i n e a n d horse transferrins. A similar two-zone p a t t e r n in bovine transferrin was observed by Tsuji et al. (1981) with the use of the same m e t h o d (mol. wts o f the two c o m p o n e n t s were 71,000 and 74,500, respectively). The two-zone p a t t e r n m a y be explained by a heterogeneity in tool. wt of transferrin. In the horse the two transferrin c o m p o n e n t s differed in their c a r b o h y d r a t e c o n t e n t s (for details see Stratil et al., 19843. The finding of the two c o m p o n e n t s in bovine transferrin is n o t in full agreement with the conclusions o f M a e d a et al. (1980), According to their interpretation, a heterogeneity in mol. wt should only a p p e a r after splitting the disulphides in the transferrin. In o u r study the two-zone p a t t e r n appeared even w i t h o u t splitting the S - - S bridges. The functional h o m o l o g y of transferrins a n d their interaction with specific receptors in all vertebrates presume t h a t the gross structure a n d c o n f o r m a t i o n of the protein (or its substantial parts) is more or less conserved. The relatively small variability in tool. wts o f transferrins o f m o s t vertebrates supports this view. W h e r e there are greater deviations (e.g. in fishes), these are a p p a r e n t l y a consequence o f changes in a part o f the molecule that is not significant for the c o n f o r m a t i o n a n d function. Acknowledgements--We wish to thank Dr. J. Williams for critical reading of the manuscript. We are indebted to Mrs M. ~,ulcovb, for skilled technical assistance. Mouse transferrin was provided by Dr. P. Dr~iber, Institute of Molecular Genetics.

REFERENCES

Aisen P., Leibman A. and Sia C.-L. (1972) Molecular wcighl and subunit structure of hagfish transferrin. Biochemistry 11, 3461-3464. Baker E., Shaw D. C. and Morgan E. H. (1968) Isolation and characterization of rabbit serum and milk transferrins. Evidence for difference in sialic acid content only. Biochemistry 7, 1371 1378. Bezkorovainy A. and Grohlich D. (19743 Comparative study of several proteins of the transferrin class. Comp. Biochem. Physiol. 47B, 787 797. Bezkorovainy A., Grohlich D. and Gerbeck C. M. (19683 Some physical-chemical properties of reduced-alkylated and sulphitolysed human serum transferrins and hen's-egg conalbumin. Biochem. J. 110, 765 770. Brock J. H., Arzabe F., Lampreave F. and Pifieiro A. (1976) The effect of trypsin on bovine transferrin and lactoferrin. Biochim. Biophys. Acta 446, 214-225. Brock J. H., Arzabe F. R., Richardson N. E. and Deverson E. V. (1978) Characterization of monoferric fragments obtained by tryptic cleavage of bovine transferrin. Biochem. J. 171, 73-..78. Buglanov A. A., Aslanov Kh. A. and Salikhov q'. A. (19803 Nekotorye fiziko-khimicheskie parametry transferrina, vydelennogo iz syvorotki krovi krys. Khim. Prir. Soedin. !, 89-92. Charlwood P. A. (1963) Ultracentrifugal characteristics of human, monkey and rat transferrins. Biochem. J. 88, 394-398. Efremov G. D., Smith L. L., Barton B. P. and Huisman T. H. J. (1971) Studies on bovine transferrin: isolation and partial characterization. Anita. Blood Grps. Biochem. Genet. 2, 159 177. Eijk H. G. van, Dijk J. P. van. Noort W. L. van, Leijnse B. and Monfoort C. H. (1972) Isolation and analysis of transferrins from different species. Scand. J. Haemat. 9, 267 270. Foucrier J., Chalumeau M.-Th. and Boffa G. A. (19763 Transferrin of the newt Pleurodeles waltlii Michah. Isolation and immunological relationship with other vertebrate transferrins. Comp. Biochem. Physiol. 53B, 555 -559. Frelinger V. A. (19733 Chemical basis of transferrin polymorphism in pigeons. Anita. Blood Grps. Biochem. Genet. 4, 35 40. Fuller R. A. and Briggs D. R. (1956) Some physical properties of hen's egg conalbumin. J. Am. Chem. Soc, 78, 5253 5257. Got R., Font J. and Goussault Y. (1967) l~tude sur une transferrine de s~lacien, la grande roussette (Scyllium stellare). Comp. Biochem. Physiol. 23, 317 327. Greene F. C. and Feeney R. E. (19683 Physical evidence for transferrins as single polypeptide chains. Biochemistry 7, 1366-1371. Gu6rin G.. Vreeman H. J. and Nguyen Y. C. (19763 Pr6paration et caractarisation physico-chimique partielle dc la transferrine s6rique ovine. Eur. J. Biochem. 67, 433~45. Hatton M. W. C., Regoeczi E., Wong K.-L. and Kra 5 G. J. (1977) Bovine serum transferrin phenotypes AA, D~D t, DzD:, EE: their carbohydrate composition and electrophoretic multiplicity. Biochem. Genet. 15, 621--.639. Hershberger W. K. (1970) Some physicochemical properties of transferrins in brook trout. Trans. Am. Fish. Soc. 99, 207 218. Hudson B. G., Ohno M., Brockway W. J. and Castellino F. J. (19733 Chemical and physical properties of serum transferrins from several species. Biochemistp~r 12, 1047 1053. Laurell C.-B. and Ingelman B. (19473 The iron-binding protein of swine serum. Acta Chem. Scand. 1, 770-776. Leibman A. J. and Aisen P. (1967) Preparation of single crystals of transferrin. Archs Biochem. Biophys. 121, 717--719.

Molecular weights of various transferrins MacGiUivray R. T. A., Mendez E., Sinha S. K., Sutton M. R., Lineback-Zins J. and Brew K. (1982) The complete amino acid sequence of human serum transferrin. Proc. Nam. Acad. Sci. USA 79, 2504--2508. Maeda K., McKenzie H. A. and Shaw D. C. (1980) Nature of the heterogeneity within genetic variants of bovine serum transferrin. Anim. Blood Grps. Biochem. Genet. 11, 63-75. Makey D. G. and Seal U. S. (1970) Serum transferrin of a reptile, the Anaconda, Eunectes murinus. Int. J. Biochem. 1, 67-76. Marneux M. (1972) l~tude de l'isotypie et de rallotypie de la transferrine chez lctalurus melas. Ann. Embryol. Morphog~n~se 5, 227-245. Morgan E. H. (1974) Transferrin and transferrin iron. In Iron in Biochemistry and Medicine (Edited by Jacobs A. and Worwood M.), pp. 29-71. Academic Press, London. Palmour R. M. and Sutton H. E. (1971) Vertebrate transferrins. Molecular weights, chemical compositions, and ironbinding studies. Biochemistry 10, 4026-4032. Richardson N. E., Buttress N., Feinstein A., Stratil A. and Spooner R. L. (1973) Structural studies on individual components of bovine transferrin. Biochem. J. 135, 87-92. Schreiber G., Dryburgh H., Millership A., Matsuda Y., Inglis A., Phillips J., Edwards K. and Maggs J. (1979) The synthesis and secretion of rat transferrin. J. Biol. Chem. 254, 12013-12019. Silberzahn P., Richard G. B. and Creyssel R. (1967) Isolement et 6tudes des protrines h proprirtrs hrmopexiques et de la transferrine du srrum de Carpe (Cyprinus carpio L.). Bull. Soc. Chim. Biol. 49, 495-506. Spooner R. L., Oliver R. A., Richardson N., Buttress N., Feinstein A., Maddy A. H. and Stratil A. (1975) Isolation

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and partial characterization of sheep transferrin. Comp. Biochem. Physiol. 52B, 515-522. Stratil A., Bob~ik P., Tom~gek V. and Valenta M. (1983a) Transferrins of Barbus barbus, Barbus meridionalis petenyi and their hybrids. Genetic polymorphism, heterogeneity and partial characterization. Comp. Biochem. Physiol. 7611, 845-850. Stratil A., Bob~,k P., Valenta M. and Tomhgek V. (1983b) Partial characterization of transferrins of some species of the family Cyprinidae. Comp. Biochem. Physiol. 74B, 603-610. Stratil A. and Spooner R. L. (1971) Isolation and properties of individual components of cattle transferrin: the role of sialic acid. Biochem. Genet. 5, 347-365. Stratil A., Tom~i~ek V., Bob~ik P. and Glasn~.k V. (1984) Heterogeneity of horse transferrin: the role of carbohydrate moiety. Anita. Blood Grps. Biochem. Genet. (in press). Tsuji S., Fukushima T., Shiomi M. and Abe T. (1981) A new serum transferrin phenotype observed in Japanese Black cattle. Anita. Blood Grps. Biochem. Genet. 12, 299-305. Valenta M., Stratil A., ~lechtov~i V., K~lal L. and ~lechta V. (1976) Polymorphism of transferrin in carp (Cyprinus carpio L.): genetic determination, isolation, and partial characterization. Biochem. Genet. 14, 27~,5. Watkins J., Tee D. E. H., Wang M. and Tarlow O. (1966) Isolation and characterization of mouse transferrins. Biochim. Biophys. Acta 127, 66-71. Webster R. O. and Pollara B. (1969) Isolation and partial characterization of transferrin in the sea lamprey, Petromyzon marinus. Comp. Biochem. Physiol. 30, 509-527. Williams J., Elleman T. C., Kingston I. B., Wilkins A. G. and Kuhn K. A. (1982) The primary structure of hen ovotransferrin. Eur. J. Biochem. 122, 297-303.