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Isolation and partial characterization of transferrin in the sea lamprey, Petromyzon marinus
Comp. Biochem. Physiol., 1969, Vol. 30, pp. 509 to 527. Pergamon Press. Printed in Great Britain
I S O L A T I O N AND PARTIAL C H A R A C T E R I Z A T I O N OF T R A N S F E R R I N IN T H E SEA LAMPREY, PETROMYZON MARINUS* R. O. W E B S T E R
a n d B. P O L L A R A
Departments of Pediatrics and Biochemistry, University of Minnesota Variety Club Heart Hospital, Minneapolis, Minnesota 55455, U.S.A. (Received 3 ffanuary 1969)
A b s t r a c t - - 1 . Transferrin was isolated and purified from sea lamprey serum by D E A E column chromatography in 0"005 M PO4 buffer, p H 8"0. 2. Lamprey transferrin has a molecular weight of 73,200-78,000 and a S20,w value of 5-5. 3. Six polymorphic forms of lamprey transferrin can be demonstrated. 4. Total amino acid analysis shows the absence of any appreciable amounts of arginine. 5. No free N-terminal amino acid was found. 6. Immunoradioautography shows the presence of two antigenically distinct iron-binding proteins.
INTRODUCTION THE TRANSFERRINSfrom blood serum have been variously called the fll metalbinding protein, siderophilin and transferrin. With ionic iron they form stable, salmon-pink complexes. In all serum transferrins examined thus far, elaborate genetic polymorphism has been demonstrated. All animals except man show a regular frequency of the different polymorphs. In humans, only one variant, type C, has appeared in high frequency in all populations (Feeney & Komatsu, 1966). The allelic forms are restricted to certain races and others only to certain kinships. No clear-cut physiologicaldifferences have been demonstrated between the various polymorphic types of transferrin. Multiple transferrins have been found by Dessauer et al. (1962) in over 150 representatives of amphibians and reptiles. Moeller& Naevdal (1966) and Creysse] et al. (1966) have found transferrin polymorphism in several different species of fish. The literature contains several other reports of polymorphism in all other classes of vertebrates (Blumberg, 1960; Arfors & Beckman, 1962; Beckman, 1962; Williams, 1962). The primary role of transferrin is that of iron transport (Hahn et al., 1942; Granick, 1946; Fletcher & Huehns, 1968). Titration and electrophoretic data (Warner & Weber, 1953; Malstrom et al., 1963; Windle et al., 1963) suggest that the binding site of iron in transferrin is made up of three phenolic groups plus bicarbonate. Two other nitrogen ligands have been implicated, either arginine, lysine or histidine or even a mixture of two of them. But, in general, the 509
510
R . O . WEBSTER AND B. POLLARA
specific metal-binding properties of the transferrins m u s t depend upon the fundamental properties of the protein as well as u p o n the positioning and reactivity of the particular ligands (Bullen et al., 1967). Cyclostomes are the most primitive living vertebrates. T o g e t h e r with the extinct Ostracoderms they f o r m the super-class Agnatha which has followed an independent evolution since earliest vertebrate origins ( R u m e n & Love, 1963). T h e only living representatives today are the hagfish and lamprey. T h e sea lamprey has been shown to have iron-binding proteins which had in the past been confused with g a m m a immunoglobulins. Boffa et al. (1967a) have called attention to the lamprey transferrins and have shown this protein to be separate f r o m the i m m u n o globulin. O t h e r studies (Finstad & Good, 1964; Papermaster et al., 1964) on the immunological capacity of the lamprey demonstrated that this animal could produce antibody responses but only to a limited n u m b e r of antigens. T h e sea lamprey appears morphologically, anatomically and biochemically to be a connecting link between the lives of invertebrates and vertebrates (Applegate, 1950; L e n h e r t et al., 1956). Because the transferrins represent a group of serum proteins with a specifically defined function, i.e. iron binding and transport, it seemed important to characterize transferrin in a primitive vertebrate like the sea lamprey with the ultimate goal of determining some pattern of evolution of the properties and structure of this important protein.
MATERIALS AND M E T H O D S A.
Experimental animals Parasitic adults and non-feeding, sexually mature adults were used in the study. All fish were maintained at the United States Bureau of Commercial Fisheries Biological Laboratory, Hammond Bay, Michigan. The parasitic adults were kept in aquaria with aerated lake water and maintained on host fish ad lib. During the spring upstream spawning migration, sexually mature adults were trapped in live boxes in the Ocqueoc River, Hammond Bay, Michigan. The adults were held in large galvanized tanks and concrete raceways with circulating lake water. Mature adults ranged in size from 30 to 60 cm with an average length of 42 cm and average weight of 200 g. B.
Collection of whole lamprey serum Individual animals were bled by cardiac puncture after anesthetizing the fish with 4-styryl pyridine. The serum was kept in the cold at 4°C or frozen at - 2 0 ° C until used. C.
Preparation of antiserum Antiserum to whole lamprey serum was prepared in both rabbits and goats. Fresh lamprey serum (0'2 ml) was mixed with an equal volume of complete Freund's adjuvant and injected subcutaneously into adult New Zealand rabbits. The rabbits were injected once a week for the first month and then once every 2 weeks. Blood was drawn from the ear vein and serum collected after centrifugation of the coagulated blood specimen. Goats were injected once a week for 2 months with 0.2 ml of whole lamprey serum. Blood was obtained via the jugular vein.
ISOLATION AND CHARACTERIZATIONOF TRANSFERRININ SEA LAMPREY
D.
511
Isolation of lamprey transferrin by column chromatography
Transferrin from the lamprey was isolated by two different methods on diethylaminoethyl (DEAE) cellulose. I n Method 1 20-25 ml of pooled, normal serum from sexually mature lampreys was dialyzed against the starting buffer (0"006 M phosphate, pH 8"0) for 16-24 hr in Visking tubing (8 ram) with constant stirring in the cold. A column (2.5 x 50 cm) was packed with 180 ml of DEAE-cellulose equilibrated with the starting buffer. T h e column was eluted in a stepwise fashion utilizing the following discontinuous gradient of phosphate buffers: 0"006 M, 0"05 M, 0"075 M, 0"1 M and 0"2 M. All buffers were at p H 8"0. Three-ml effluent fractions were monitored at 280 m/z on a Beckman D U spectrophotometer. T h e absorption peaks were pooled and concentrated by means of positivepressure pervaporation. The concentrated pools were then dialyzed against the initial buffer to remove salts. Method 2 incorporated the use of a continuous gradient as outlined by Peterson & Sober (1959) modified by Bridges (personal communication). Serum from individual fish was labeled with iron-59. The labeled serum was placed on a small column (2.2 x 25 cm) after dialysis in the cold with constant stirring for 16-24 hr against the starting buffer to remove u n b o u n d iron-59. Fifty ml of each of the following buffers was used in each chamber: 1. 0"005 M phosphate, pH 8"0 2. 0"03 M phosphate, pH 7"88, 0"004 M NaC1 3. 0'06 M phosphate, p H 7"74, 0'003 M NaCI 4. 0"06 M phosphate, pH 7'59, 0-032 M NaCI 5. 0"06 M phosphate, p H 7.44, 0"061 M NaC1 6. 0'06 M phosphate, pH 7"29, 0"090 M NaC1 7. 0"06 M phosphate, pH 7"15, 0'119 M NaC1 8. 0"06 M phosphate, p H 7"00, 0"150 M NaC1 9. 0'06 M phosphate, pH 6"00, 0-300 M NaC1 T h e fractions were monitored at 280 m/z on the Beckman D U and were pooled and concentrated by pervaporation and later dialyzed against the starting buffer.
E.
Radioisotope labelling of serum
Fifteen/zc of iron-59 citrate solution were added per ml of serum or isolated transferrin fraction. The mixture was dialyzed in Visking cellulose tubing (8 ram) against multiple changes of 0"005 M phosphate buffer, pH 8"0. The dialysis was carried out for 16-20 hr in the cold with constant stirring to remove excess iron atoms.
F.
Microimmunoelectrophoresis
Immunoelectrophoresis was carried out as described by Scheidegger (1955) modified by Bridges (personal communication) using a gel containing 1"5 ~o (w/v) agar.
G.
Protein analysis
Relative protein concentrations were determined by measurement of the absorbance at 280 rn/z. More accurate protein concentrations were determined by the method of Folin & Ciocalteu (1927) as modified by Lowry et al. (1951). An alternative method for the spectrophotometric determination of protein concentration was the method of T o m b s et al. (1959).
H.
Vltracentrifugation analysis 1. Sedimentation velocity. Sedimentation velocity studies were carried out in the Spinco
Model E analytical ultracentrifuge. A 12-rnm wedge window, single-sector aluminum cell was used at a rotor speed of 59,780 rev/min. The temperature was maintained constant at 20°C by means of a R I T C unit. Schlieren optics were used with a red filter (655 m/z) and
512
R . O . WEBSTERAND B. POLLARA
Kodak spectroscopic plates (Type I-N). Sedimentation coefficients were calculated from the equations given by Schachman (1959). The S~0,w values at zero concentration was obtained by graphical extrapolation of the calculated S2o,w values. 2. Molecular weight determination (by ultracentrifugation or column chromatography). The molecular weight of transferrin was determined in the analytical ultracentrifuge at 20°C by the high-speed sedimentation equilibrium method using interference optics described by Yphantis (1964). Sapphire windows were used at all speeds and protein concentrations between 0'8 and 3.6 mg/ml were employed. A green filter (546 rn/~) was used. Kodak spectroscopic glass plates (Type II-G) were measured in a Nikon comparator after which the weight-average molecular weight was calculated by the least-squares fit of a In C vs. r ~ plot. The Z-average molecular weight was calculated from the equation of Yphantis (1964). Calculations were made with the aid of a Control Data Corporation 3300 computer using the Fortran program of Small & Resnick. The molecular weights of several transferrin samples were estimated by the column chromatography method of Andrews (1964) and Leach & O'Shea (1965) modified by Bridges (personal communication). An upward-flowing, temperature-controlled column (3"2 × 80 cm) was filled with G-200 Sephadex. One- to 2-ml samples were placed on the column at regular intervals so that no overlapping of protein peaks would occur. All of the runs were made at 4°C. Cytochrome-c, albumin and glutamate dehydrogenase were used as calibration standards. The column effluent was monitored at 210 m/z to establish the position of the protein peaks. The column volumes were estimated by pooling the collected fractions.
I.
N-terminal amino acid analysis Two methods were used for N-terminal amino acid analysis. The dansyl technique was used because of the small quantities required (m/zmoles) while the Edman method was used because of the ease of preparation and identification of the N-terminal amino acid derivatives. 1. The dansyl method. The dansyl reagent [1-dimethylaminonaphthalene-5-sulfonyl chloride (DNS-CI)] was used as suggested by Gray & Hartley (1963). Standard D N S amino acids were purchased from Gallard-Schlesinger (N.Y.). Chromatographic identification was achieved by the method of Woods & Wang (1967). 2. The modified Edman method. The Edman method (1953, 1960), as modified by Sjoquist (1957) and Blomback et al. (1966), was initially used to try to determine a short sequence of N-terminal amino acids. In each case 5/~moles of protein were used. The P T H amino acids were chromatographed using the method of Jeppsson & Sjoquist (1957). P T H amino acid standards were purchased from the Mann Research Laboratory, Inc., N.Y. Eastman chromagram sheets type K301R with fluorescent indicator were activated at 105°C 30 rain before use. Each side of a sheet in the direction of a chromatography was scrapped 1 cm wide in order to prevent front-line convection. The samples were applied 1"5 cm apart under a stream of nitrogen. J.
Amino acid analysis Total amino acid analyses were performed according to the method of Moore et al. (1958) and Spackman (1962) in a Spinco automatic amino acid analyzer Model 120B. The acid hydrolysis was carried out in 6 N HC1. The results were obtained in/zmole per cent, and converted to residues per mole of transferrin with the following equation : /zmoles amino acid X M.W. transferrin (rag) residues 1 mg transferrin 1 m-mole transferrin mole K.
Sialic acid assay The sialic acid content was determined by the thiobarbituric acid method described by Warren (1959).
ISOLATION AND CHARACTERIZATION OF TRANSFERRIN I N SEA LAMPREY
513
L. Polyacrylamide disc-electrophoresis Transferrin and other proteins were qualitatively separated by disc-electrophoresis involving the use of 7% polyacrylarnide gel as described by Ornstein (1964) and Davis (1964) modified by Hong (personal communication).
M. lmmunoradioautographs Radioautographs of the dipping type were done according to the method of Messier & Leblond (1957). Immunoelectrophoresis of the desired samples was carried out as previously described. Fifteen /zc of iron-59 were added to 0"2 ml whole lamprey serum and the mixture dialyzed against 0"005 M phosphate, pH 8"0, to remove unbound iron-59. The slides were developed for 7-14 days. RESULTS
A.
Isolation of pure lamprey transferrin
Stepwise elution of the DEAE-cellulose column with five buffers of increasing ionic strength (0.006-0.2 M phosphate, p H 8.0) resulted in five protein peaks of which the first peak contained the isolated transferrin. Careful attainment of column equilibrium with the starting buffer resulted in isolation of pure transferrin. Purity was checked by immunoelectrophoresis and by analytical ultracentrifugation. A representative chromatogram of whole lamprey serum separation can be seen in Fig. 1. DEAE
WHOLE LAMPREY SERUM
200
I
I. 6 0
OD2Bo 1.20
8
I
30
7O
I10
150
Fraction No.
FIG. 1. Chromatogram of pooled whole lamprey serum on DEAE-cellulose using step-wise elution. All buffers at pH 8'0. Separation of individual lamprey serum labeled with iron-59 was effected by the use of a small DEAE-cellulose column and a continuous gradient for elution.
514
R. O. WEBSTrRAND B. POLLARA
The gradient was not (transferrin) was eluted spontaneous release of serum separation using DEAE
applied to the column until after the first protein peak from the column. Use of a continuous gradient minimized radioactivity. A typical chromatogram of whole lamprey a continuous gradient is shown in Fig. 2.
WHOLE
LAMPREY
SERUM CONTINUOUS GRADIENT
1.40
7O
1.20
6O
1.00
5O
.80
4O
CPM
X[O 2
ODzeo o--.,o
.60
50
401
2O
]0
__
20
25
60
70
1 0
l
80 Frection
i I00
IIO
No.
Fic. 2. Chromatogram on DEAE-cellulose of whole lamprey serum using a continuous gradient of 0"005 M PO4, pH 8"0-0"06 M PO4 with 0'300 M NaCI. Those samples of transferrin containing iron-59 were placed on an Andrew's column containing G-200 Sephadex in order to estimate the molecular weight. Figure 3 shows a typical chromatogram in which the radioactive peak corresponds directly to the protein peak of transferrin. Analysis of the isolated transferrin samples by microimmunoelectrophoresis showed that the lamprey transferrin migrates to the cathode as Boffa et aL (1967a) had previously described. B.
Protein analysis
The spectrophotometric method of Tombs et al. (1959) proved to be the method of choice in determining the protein concentration of both lamprey transferrin and serum. The most noticeable fact in the measurement of total serum proteins in the lamprey is the large decrease in protein concentration as the animal matures. Quantitative experiments showed that the total serum protein level varied from 74.0 to 34.6 mg/ml in the parasitic adults and from 70-2 to 21.1 mg/ ml in the sexually mature adults. This represents a possible decrease of as much
ISOLATION AND CHARACTERIZATION OF TRANSFERRIN IN SRA LAMPREY
515
as 71.4 per cent. T h e highest concentration of pure serum transferrin isolated from one individual fish was 4.95 mg or 9"3 per cent of the total serum protein. Tronsferrin - Fe 59 on Sephodex G - 2 0 0 O.06M PO4 ~" 0.075M KCI
lO0
.800
8O !
ODzto .6oo ¢.-,-..o
60
.400
40
200
20
260
I
I
T
270
280
290
CPM XlO z
Froction No.
FIC. 3. Chromatogram of transferrin labeled with iron-59 on G-200 Sephadex (0"06 M PO4 with 0"075 M KC1).
C.
Ultracentrifugation studies 1. Sedimentation velocity. Sedimentation velocity analyses of transferrin in
varying concentrations were carried out in 0.005 M phosphate buffer, pH 8.0. Sedimentation values and concentration are listed in Table 1. Concentrations of transferrin were plotted against sedimentation values corrected to standard conditions (Fig. 4). At infinite dilution the S~o,w value was 5.5. TABLE l m S U M M A R Y OF Sso,t o VALUES FOR TRANSFERRIN (DETERMINED AT ROTOR SPEED OF
59,780 rev/min) Cone. (mg/ml)
S~o,w
1.12 1.71 4.05 6.11 7-50
5.42 5.37 5-25 5.09 5-01
2. Molecular weight determination. Equilibrium molecular weights of transferrin were obtained with the multichannel cell of Yphantis for three different concentrations of transferrin at two speeds. T h e runs were made in phosphate
516
R. O. WEBSTER AND B. POLLARA TRANSFERRIN
SPEED
59.780
S20,W
501 I
I
i L__
I_
I0
I
2.0
1
30
I ___i
40
50
I
6.0
I
ZO
8.0
Oonc. mg/ml
Fro. 4. Relation of S2o,w to concentration of transferrin.
-251
I i
-2 7 !
ii -5. r ~ I
J -351
I
InO
-3.9~ - 4 3~~ ' ~ * / I i
-47[ i I[____J___ 600
42550
.700
I
.800
__ I
900
.__~
43000
I
.~6o
£
.200
J
.300
L 4~00
.500
Radius Squared
Fzc. 5. Relationship of natural logarithim of concentration to radius squared showing homogeneity and ideal conditions.
ISOLATION AND CHARACTERIZATION OF TRANSFERRIN IN SEA LAMPREY
517
buffer (0-005 M, pH 8-0) at 20°C. The average molecular weight of the protein was 73,200 + 2200, assuming a partial specific volume, ~ of 0.73. A representative plot of In concentration vs. radius squared is shown in Fig. 5. Examination of the plot of the logarithim of the concentration vs. the radius squared yields evidence of the degree of homogeneity or nonideality. As "Small & Lamm (1966) point out the nonlinearity of the plot where the plot has a slight but definite upward curvature is an indication of heterogeneity, whereas a downward curvature is an indication of nonideal conditions. The determination shown in Fig. 5 approaches ideal conditions and homogeneity.
D.
Molecular weight determination by column chromatography
The molecular weight of transferrin was also determined by use of an Andrew's column in addition to the high-speed equilibrium method of Yphantis. The standards and their respective molecular weights used in the calibration of the column are as follows: cytochrome-c, 12,400; albumin, 67,000; and glutamate dehydrogenase, 250,000 (Andrews, 1964). The column volumes of the standards and of several different transferrin fractions are given in Table 2. The standard curve from which the molecular weight of transferrin was obtained is shown in Fig. 6. The molecular weight of transferrin by this method was estimated as 78,000. TABLE 2--CoLuMN
Average
VOLUMES OF K N O W N STANDARDS AND LAMPREY TRANSFERRIN RUN ON A N D R E W ' S COLUMN (VALUES I N ml)
Cytochrome-c
Albumin
Glutamate dehydrogenase
525 540 528 535 519 523 543
338 356 343 345 330 334 346
229 236 226 225 222 222 223
530
342
226
Transferrin
Estimated weight
336 337 337 334
79,000 78,000 78,000 80,000
336
78,750
Average
E. Amino acid composition The amino acid composition of lamprey transferrin is given in Table 3. Tryptophan and cysteine, destroyed by acid hydrolysis, were not measured due to the
518
R. O. WEBSTERAND B. POLLARA
large amounts of protein needed for reaction. Since the amide forms of the dicarboxylic amino acids were converted to the acid forms upon acid treatment, glutamic acid and glutamine were reported together, as were aspartie and asparagine. Only a slight trace of arginine was found to be present in the pooled sample of transferrin. STANDARD CURVE ANDREWS COLUMN I00 .80 60
40
Glulomo/e Oehydrogenose 20
MWxlO 4
I0
Tronsferr/n
8 6
Cyfochrome C I
I
1
L~
~
T
I00
200
~00
400
500
Effluent
Volume .(ml)
FIG. 6. Standard elution curve from Andrew's column (G-200 Sephadex, 0"06 M PO4 with 0"075 M KC1, pH 8"0).
F.
Sialic acid assay
Three different samples of isolated transferrin were subjected to the sialic acid assay of Warren (1959). Standards made up of pure N-acetyl neuraminic acid were run at the same time as the isolated transferrin samples. In every case, there were no residues of sialic acid detected.
G.
N-terminal amino acid analysis and sequence analysis Experiments to determine the N-terminal amino acid of transferrin were done by two methods. Using the Edman method, the first or N-terminal residue did not migrate with either solvent system used, suggesting that the N-terminal was
ISOLATIONAND CHARACTERIZATIONOF TRANSFERRININ SEA LAMPREY
519
cysteic acid. An attempt was m a d e to couple and cleave off the second amino acid residue, T h i s yielded a similar spot. TABLE 3 - - A M I N O ACID COMPOSITION OF LAMPREY TRANSFERRIN*
Trial 1
Asp Thr Ser Glut Prol Gly Ala Val Met Ile Leu Tyr Phe Lys His Arg
Trial 2
Trial 3
Res/
Res/
Res/
Res/
Res/
Res/
73,200 g
10,000 g
73,200 g
10,000 g
73,200 g
10,000 g
38.4 22"2 32"0 29"2 29'2 22"6 61"8 39"2 12"8 23"2 39"6 12'0 25"4 49.8 6.6 Trace
5.2 3"0 4"4 4"0 4"0 3"0 8"4 5'4 1"8 3'2 5"4 1"6 3"4 6.8 1.0
57.2 35"8 49-8 42"0 29"0 36"8 81"2 49"8 15"8 22"2 40"8 18"8 28"2 --~ . .
7.8 5'0 6"8 5"8 4"0 5"0 11-2 6"8 2'2 3'0 5"6 2"6 3"8 --
39.8 24"0 34"2 29-2 18'4 24"8 55"8 33"0 9"8 15'8 27"6 10"4 20"8 --
5.4 3"2 4"8 4'0 2"6 3'4 7"6 4-6 1"4 2"2 3"8 1'4 2"8 --
. .
. .
. .
* Determination did not account for tryptophan, cysteine, iron content nor carbohydrate content. Not determined. Since b o t h of these derivatives behaved in this manner, it was thought that the spot could represent degradation products. Serine and threonine, usually destroyed by acidic conditions, were checked for b y using the alternative E d m a n m e t h o d (Guidotti et al., 1962). No P T H residues were found u p o n c h r o m a t o g r a p h y of the material obtained f r o m the reaction mixture indicating that the N - t e r m i n u s might be blocked. Since an N - t e r m i n a l group could not be detected using the E d m a n method, the highly sensitive dansyl m e t h o d was employed. I t was impossible to obtain fluorescent spots f r o m the transferrin reaction mixture corresponding to the labelled amino acid standards suggesting that the N - t e r m i n u s was indeed blocked (possibly by an aeetyl group or pyrrolidone carboxylic acid).
H.
Polyacrylamide disc-electrophoresis
U p o n examining isolated and purified transferrin fractions by polyacrylamide gel discs it was seen that as m a n y as six different transferrin bands were found in a pooled sample (Fig. 7). T h i s suggested the presence of six differently charged transferrin species. W h e n the gels were sliced horizontally and counted in a g a m m a
520
R . O . WEBSTER AND B. POLLARA
counter, radioactive peaks obtained coincided exactly with the protein bands revealed by staining. Thus, specific iron-binding capacity was present in all six bands. Transferrin fractions obtained from individual animals and labeled with iron-59 were also placed on microdisc-electrophoresis where only one band was found. I.
Immunoradioautographs The radioautographs of the isolated transferrin samples from sexually mature adults showed a single line of gamma mobility. On the other hand, those immunoradioautographs done on whole lamprey serum showed two different patterns. Whole serum from sexually mature adults (Fig. 8) showed the same single band as the sample of isolated and purified transferrin. However, whole serum from the parasitic type adults (Fig. 9) contained two iron-binding proteins. These two iron-binding proteins were antigenically distinct as the protein arcs crossed each other. There was a homogeneous, fast migrating band and one very heterogenous, slow migrating band.
DISCUSSION In several reports (Boffa et al., 1966, 1967a, b), it has been suggested that lamprey transferrin migrates in the area of gamma immunoglobulins as shown on immunoelectrophoresis. Subsequently, it has been seen (Pollara, unpublished observation) that the immunoglobulins in this animal have alpha or fast beta mobility as shown on microimmunoelectrophoresis. It has also been demonstrated, by use of the functional properties of transferrin binding iron-59 and immunoglobulin precipitating Brucella antigen, that these proteins possess distinctly different electrophoretic mobilities. A.
The nature of transferrin in the sea lamprey In many respects lamprey transferrin does not seem to differ widely from the other characterized transferrins. The molecular weight of lamprey transferrin (73,200-78,000) is like the transferrins of carp (70,000) (Silberzahn et al., 1967) and human (73,000-78,000) (Roberts et al., 1966). There is a difference in the value obtained by ultracentrifuge method (73,200) and the value obtained by the Andrew's columns (78,000). A partial specific volume (17) was assumed to be 0.73, based on reported values for other transferrins (Putnam, 1965). This assumption may account for the discrepancy in the molecular weights. The corrected sedimentation velocity value of 5"5 is slightly higher than the value given by Boffa et al., (1967a, b) of 5.1. Roberts et al. (1966) described the dependence of sedimentation velocity of human transferrin on pH. Boffa's data did not include conditions which were used to perform the ultracentrifuge experiments. Hence, no comparison can be made between the different sets of data. The dependence of the sedimentation velocity of lamprey transferrin on pH was not investigated in this study.
+ FIG. 7. Disc-electrophoresis of isolated transferrin obtained from pooled serum (anode at bottom).
FIC. 8. Immunoradioautographs of sexually mature lamprey serum tagged with iron-59 (15 /zc/rnl of serum). Whole lamprey serum in antigen wells with goat anti-whole lamprey serum in antibody trough.
L
FIG. 9. Immunoradioautographs of whole parasitic lamprey serum tagged with iron-59 (15/zc/ml of serum). Lamprey serum in antigen wells with goat anti-whole lamprey serum in antibody trough.
ISOLAT~ONANDCHARAC~mZATIONOr ~ANSFrRmNm SEALA~P~
521
The absence of any appreciable amount of arginine in the total amino acid analysis provides considerable evidence that this amino acid is excluded from consideration as one of the possible N-ligands of the binding site of iron in transferrin. Buttkus et al. (1965) have concluded from blocking studies that "if two nitrogen ligands are directly involved in metal binding, these nitrogens are probably not contributed to by the e-amino groups of lysine". This would leave only histidine as the likely amino acid in the role of N-ligand in lamprey transferrin. This would be entirely comparable to the situation as it is found in the binding site of oxygen in hemoglobin. N-terminal amino acid analysis failed to reveal an active N-terminal amino acid. Based on an estimated molecular weight of 70,000, an adequate amount of protein was used in these determinations. Absence of a reactive amino acid suggests that the N-terminus may be blocked by an acetyl group or pyrrolidone carboxylic acid. The presence of these groups as N-terminals has been seen in a number of other proteins (Press et al., 1966; Putnam et al., 1967). The reduction of the serum protein level As the lamprey matures beyond the parasitic stage, a marked decrease occurs in the serum proteins. Uthe & Tsuyuki (1967), using polyacrylamide disc-electrophoresis of blood proteins of adult and ammocete lampreys, showed a significant decrease in the total amount of protein. Rall et aL (1961) showed that the alpha globulins decrease from 70 per cent in the larval forms to 37 per cent in the mature adult. In our quantitative protein study, the total serum protein levels were seen to decrease over 70 per cent as the lamprey matures from the parasitic stage to the sexually mature stage. As Thomas & McCrimmon (1964) point out, the parasitic lamprey represents a rapid period of growth in the life history of the sea lamprey in the open lake which lasts for 12-18 months. During the spring migration which follows the parasitic feeding stage, lamprey ascend tributary streams to spawn. The lamprey do not feed during the spawning migration and die following the spawning process. Applegate (1950) has demonstrated morphologically that a severe involution of the digestive tract takes place once feeding has stopped. Thus, it seems that the serum proteins are used as metabolites and basic building blocks as the body requires them. Since there is no other means of obtaining any foodstuffs, the serum could be a primary protein source resulting in a decreasing protein concentration as the animal ages. B.
C.
Polymorphism in lamprey transferrin The elaborate polymorphism of transferrin found in many other vertebrate animals is also found in the sea lamprey. Boffa et al. (1967a) reported finding five transferrin bands in the lamprey by the method of radioautography after electrophoresis on starch-gel blocks and paper strips. Since several authors (Ornstein, 1964; Rausch et al., 1965; Margolis & Kenrick, 1967) mentioned that fine protein 'separations are posisble on polyacrylamide gel, it was elected to use this method using transferrin labeled with iron-59. These studies showed the existence of six
522
R . O . WEBSTER AND B. POLLARA
different transferrin bands. When the gels were sliced into equal parts and counted in a radiation counter, the radioactive peaks corresponded directly to the six stained protein bands. Parker & Beam (1961a, b) found that starch-gel electrophoresis of labeled human transferrin C - - C mixed with neuraminidase from Vibrio cholerae revealed a step-wide pattern of five bands whose intensities varied with neuraminidase concentration. They attributed the different migrating species to the amount of sialic acid attached to the transferrin. The fastest moving band coincided with untreated transferrin and contained approximately four sialic acid residues per molecule o5 protein. On the other hand, the slowest moving band represented complete removal of sialic acid. Subsequent analysis of other transferrin variants with different migration rates showed that they all contained four sialic acid residues per molecule of transferrin. So it became apparent that the difference in migration was not due to the protein having less than four sialic acid residues. The absence of sialic acid residue in lamprey transferrin indicates that the observed differential rates of migration would have to be due to the presence of different electric charges on the protein. This can be accounted for by substitution of different amino acids within the protein. Such substitutions could be controlled by alterations of the genome directing transferring synthesis. The results suggest a hypothesis; that the genetic polymorphism shown here may be controlled by three alleles, A, B and C. The six variants could be made up of the following genotypes: AA, BB, CC, AB, AC and BC. In a review article, Giblett (1962) mentioned that Smithies & Hiller (1959) suggested the symbol Tffor the autosomal locus with the superscript letters for the type of protein variant. Giblett further states that the fact that no human serum has been conclusively shown to contain three transferrins provides some indirect evidence that the transferrin variants represent a series of alternative dominant alleles at a single locus. Such also could be the case in the sea lamprey. It is doubtful that the variants are due to varying degrees of aggregation of polymer types of structure. If such were the case, it would have been detected in the Yphantis equilibrium run. There would also have been more than one peak observed while doing the sedimentation velocity experiments. Furthermore, the high order of sensitivity using iron-59 labeled transferrin on G-200 Sephadex is good evidence favouring monomer over polymer types of structure.
D.
Two iron-binding proteins in the sea lamprey One of the least expected discoveries in this study was the presence of two antigenically distinct iron-binding proteins in the lamprey. To further complicate the picture, the two iron-binding proteins were found only in the parasitic animals and not in the sexually mature animals. The immunoradioautographs show a complete immunological non-identity between the two proteins. The slower migrating, heterogeneous band eluted as the first peak on DEAE chromatography. This was identified as the lamprey transferrin. The fast, migrating homogeneous peak eluted with the last half of the fourth protein peak. Only the transferrin was
I S O L A T I O N AND CHARACTERIZATION OF TRANSFERRIN I N SEA LAMPREY
523
chromatographically pure as shown by microimmunoelectrophoresis. The second iron-binding protein was contaminated with several of the alpha-migrating proteins. No attempt was made to isolate this protein due to the extremely small amounts of protein available. The presence of two types of iron-binding proteins in other animals has been mentioned only briefly. Reports have been made involving rats (Gordon, 1963; Gordon & Louis, 1963), hagfish (Manwell, 1963) and man (Shirasawa, 1965). Only in the latter case has any investigation been done. Shirasawa separated these two components of transferrin on TEAE-cellulose chromatography but did not isolate the second component. He estimated that the minor component of transferrin C - - C comprised about 5-10 per cent of the radioactivity of the major component. The radioactivity of the second lamprey iron-binding protein was about 35 per cent of the transferrin peak, although this may merely reflect differences in iron-binding capacity. Lai & Kirk (1960) and Sutton & Bowman (1961) have reported a monkey with three iron-binding proteins. It has not been determined yet whether this phenomenon is due to a single gene determining more than one protein band or the presence in some monkeys of more than one transferrin locus. The disappearance of the fast migrating, homogeneous band in the sexually mature animal could be accounted for in two ways. The first possibility is that the fast migrating form is an early precursor form of transferrin which undergoes a transformation to one of the six allelic variants of the slower migrating form of transferrin. The other possibility is that upon the involution of the lamprey's digestive system, this protein is expendable and is used for nutritional purposes. SUMMARY Lamprey transferrin was isolated from whole serum by means of DEAE column chromatography. The isolated transferrin has been studied by means of immunological, physical and chemical methods. The purification of lamprey transferrin was accomplished by the elution of protein from a DEAE-cellulose column with the starting buffer (0.005 M phosphate, pH 8-0). Gel electrophoresis of the isolated transferrin fraction showed six electrophoretically different migrating bands. These polymorphs could be explained by the presence of three allelic autosomal genes of equal dominance. Quantitative analysis of the total serum proteins of the lamprey show a marked decrease as the lamprey ages from the parasitic to the sexually mature stage. This decrease ranges up to 70 per cent of the peak found in the parasitic fish. Upon reaching sexual maturity, the digestive tract of the lamprey involutes so that no further feeding of the fish can take place. Hence, the serum proteins are thought to be used as metabolites in required body functions. Immunoradioautography of parasitic lamprey serum reacted with iron-59 shows two antigenically distinct iron-binding proteins. One is a fast, migrating, homogeneous band and the other a slower migrating, heterogeneous band. The slower migrating band is the one that was isolated on DEAE chromatography and characterized. On sexual maturation, the fast migrating iron-binding protein
524
R. O. WEBSTERAND B. POLLARA
disappears f r o m the serum suggesting a possible functional difference in these proteins. Ultracentrifuge studies show a S~0, w value of 5.5 and a molecular weight of 73,200 + 2200. Estimation of the molecular weight on G-200 Sephadex chromatography gave an average value of 78,000. T h e absence of any sialic acid residues was confirmed. Reaction with the E d m a n and dansyl reagents failed to demonstrate a free N - t e r m i n a l amino acid suggesting that the N - t e r m i n a l residue is blocked. Total amino acid analysis shows the absence of arginine, thus excluding this amino acid f r o m participating in the binding site of iron and indicating histidine as the probable nitrogen ligand in the binding site. Acknowledgements--We would like to thank Dr. Robert A. Bridges for many helpful discussions and suggestions, and Mr. John H. Howell for his co-operation in maintaining the lamprey at Hammond Bay. Aided by grants from the National Foundation and U.S. Public Health Service (AI-08372).
REFERENCES ANDREWS P. (1964) Estimation of the molecular weights of proteins by Sephadex gelfiltration. Biochem. ff. 91, 222-233. APPLEGATE V. C. (1950) Natural history of the sea lamprey. U.S. Dept. Interior, Special Sci. Rep. Fisheries 55, 1-237. ARFORS K. E. & BECKMANL. (1962) Phylogentical and immunological aspects of primate serum transferrins. Hereditas 48, 132-137. BECKMAN L. (1962) Slow and fast transferrin variants in the same pedigree. Nature, Lond. 194, 796-797. BLOMBACK B., BLOMBACK M., EDMAN P. •
HESSEL B. (1966) H u m a n fibrinopeptides,
isolation, characterization and structure. Biochim. biophys. Acta 115, 371-396. BLUMBERG B. S. (1960) Biochemical polymorphisms in animals: haptoglobins and transferrins. Proc. Soc. exp. Biol. Med. 104, 25-28. BOFFA G. A., FINE J. M., DRILHON A. 8¢ AMOUCH P. (1967a) Irnmunoglobutins and transferrin in marine lamprey sera. Nature, Lond. 214, 700-702. BOFFA G. A., FINE J. M. &; FAURE A. (1967b) NouveUes donn6es sur la transferrine et les immunoglobulines de la lamproie marine. Bull. Soc. Chim. Biol. 49, 433-445. BOFFA G. A., MARINELLIG., DBILHONA. & FINE J. M. (1966) Evidence for and isolation of transferrin in the marine lamprey. C.r. hebd. Sdanc. Acad. Sci., Paris 262, 2294-2297. BULLEN J. J., ROGERS H. J. ~% CUSHNIE G. H. (1967) Abolition of passive immunity to bacterial infections by iron. Nature, Lond. 214, 515-516. BUTTKUS H., CLARK J. R. & FEENEY R. E, (1965) Chemical modifications of amino groups of transferrins: ovotransferrin, human serum transferrin and human lactotransferrin. Biochemistry 4, 998-1005. CREYSSEL G., RICHARD G. B. & SILBERZAHNP. (1966) Transferrin variants in carp serum.
Nature, Lond. 212, 1362. DAVIS B. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Annls. N . Y . Acad. Sci. 121, 404-427. DESSAUER H. C., Fox W. & HARTWIG Q. L. (1962) Comparative study of transferrins of Amphibia and Reptilia using starch-gel electrophoresis and autoradiography. Comp. Biochem. Physiol. 5, 17-29.
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EDMAN P. (1953) Selective Cleavage of Peptides in the Chemical Structure of Proteins (Edited by WOLSTENHOLME G. E. W. & CAMERON M. P.), pp. 98-101. Ciba Foundation Symposium, Boston. EDMAN P. (1960) Phenylthiohydantoins in protein analysis. Annls. N.Y. Acad. Sci. 88, 602-610. FEENEY R. E. & KOMATSU S. K. (1966) The Transferrins in Structure and Bonding (Edited by JORGENSEN C. K., NEILANDSJ. B., NYHOLM R. S., REINEN D. & WILLIAMS R. J. P.), Vol. 1, pp. 149-206. Springer, New York. FINSTAD J. & GOOD R. A. (1964) T h e evolution of the immune r e s p o n s e - - I I I . Immunologic responses in the lamprey, ft. exp. Med. 120, 1151-1168. FLETCHER J. & HUEHNS E. R. (1968) Function of transferrin. Nature, Lond. 218, 1211-1214. FOLIN O. & CIOCALTEU V. (1927) On tyrosine and tryptophane determination in proteins. ft. biol. Chem. 73, 627-650. GIBLETT E. R. (1962) Plasma transferrins. Prog. Med. Genet. 2, 34-63. GORDON A. H. (1963) The rates of catabolism of rat transferrin in vivo and by the perfused rat liver. Protides Biol. Fluids, Proc. Colloq. 10, 71-73. GORDON A. H. & LouIs L. N. (1963) Preparation and properties of rat transferrin. Biochem. J . 88,409-414. GRANICK S. (1946) F e r r i t i n - - I X . Increase of the protein apoferritin in the gastrointestinal mucosa as a direct response to iron feeding. The function of ferritin in the regulation of iron absorption. J. biol. Chem. 164, 737-746. GRAY W. R. & HARTLEY B. S. (1963) A fluorescent end-group reagent for proteins and peptides. Biochem. J. 89, 59P. GUIDOTTI G., HILL R. J. & KONIGSBERC W. (1962) T h e structure of human h e m o g l o b i n - II. T h e separation and amino acid composition of the tryptic peptides from the alpha and beta chains. J. biol. Chem. 237, 2184--2195. HAHN P. F., Ross J. F., BALE W. F., BALFOUR W. M. & WHIPPLE G. H. (1942) Red cell and plasma volumes (circulating and total) as determined by radio iron and by dye. J. exp. Med. 75, 221-232. JEPPSSON J. O. & SJOQUIST J. (1967) Thin layer chromatography of P T H amino acids. Mnalyt. Biochem. 18, 264-269. LAI L. Y. C. & KIRK R. L. (1960) Beta-globulin variants in two species of monkeys. Nature, Lond. 188, 673-674. LEACH A. A. & O'SHEA P. C. (1965) T h e determination of protein molecular weights of up to 225,000 by gel filtration on a single column of Sephadex G-200 at 25 ° and 40 °. J . Chromatogr. 17, 245-251. LENHERT P. G., LOVE W. E. & CARLSON F. D. (1956) T h e molecular weight of hemoglobin from Petromyzon marinus. Biol. Bull., Woods Hole 111, 293-294. LOWRY O. H., ROSEBROUCHN. J., FARE A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MALSTROM R., VANNGARDT., AASA R. & SALTMANP. (1963) On the nature of the metalbinding sites in transferrin and conalbumin. Fedn Proc. 22, 595. MANWELL C. (1963) The blood proteins of cyclostomes. In A Study in Phylogenetic and Ontogenetic Biochemistry in the Biology of the Myxine (Edited by BROOALA. & FANGE R.), pp. 237-455. Universitetsforlaget, Oslo. MARGOLISJ. & KENRICK K. G. (1967) Polyacrylamide gel-electrophoresis across a molecular sieve gradient. Nature, Lond. 214, 1334-1336. MESSIER n. & LEBLOND C. P. (1957) Preparation of coated radioautographs by dipping sections in fluid emulsion. Proc. Soc. exp. Biol. Med. 96, 7-10. MOELLER D. & NAEVDALG. (1966) Serum transferrins of some gadoid fishes. Nature, Lond. 210, 317-318. MOORE S., SPACKMAN D. H. & STEIN W. H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. An improved s y s t e m . . 4 n a l y t . Chem. 30, 1185-1190. i8
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ORNSTEIN L. (1964) Disc electrophoresis--I. Background and theory. Annls. N.Y. Acad. Sci. 121, 321-349. PAPERMASTER B. W., CONDIE R. M., FINSTAD J. &; GOOD R. A. (1964) Evolution of the immune response--I. T h e phylogenetic development of adaptive immunologic responsiveness in vertebrates, ft. exp. Ned. 119, 105-130. PARKER W. C. &; BEARNA. G. (1961a) Variations in sialic acid content of human transferrins. Fedn Proc. 20, 255. PARKERW. C. & BEARNA. G. (1961b) Alterations in sialic acid content of human transferrin. Science 133, 1014-1016. PETERSON E. A. & SOBERH. A. (1959) Variable gradient device for chromatography. Analyt. Chem. 31, 857-862. POLLARA B. (1967) Unpublished observation PRESS E. M., GlVOL D., PIGCOT P. J., PORTER R. R. & WILKINSON J. M. (1966) The chemical structure of the heavy chains of rabbit and human immunoglobulin G (IgG). Proc. R. Soc. (Lond.) B 166, 150-158. PUTNAM F. W. (1965) Structure and function of the plasma proteins. In The Proteins (Edited by NEURATH H.), Vol. 3, pp. 153-267. Academic Press, New York. PUTNAM F. W., SHINODA T., TITANI K. & WIKLER M. (1967) Immunoglobulin structure: variation in amino acid sequence and length of human light chains. Science 157, 10501053. RALL P. P., SCHWAHP. & ZUBaOD G. (1961) Alteration of plasma proteins at metamorphosis in the lamprey (Petromyzon marinus dosatoss). Science 133, 279-280. RAUSCH W. H., LUDWlCK T. M. & WESELI D. F. (1965) Determination of bovine transferrin types by disc electrophoresis. J. Dairy Sci. 48, 720-725. ROBERTS R. C., MAKEY D. G. & SEAL U. S. (1966) Human transferrin--molecular weight and sedimentation properties. J. biol. Chem. 241, 4907-4913. RUMEN N. M. & LOVE W. E. (1963) The six hemoglobins of the sea lamprey (Petromyzon marinus). Archs Biochem. Biophys. 103, 24-35. SCHACHMAN H. K. (1959) Ultraeentrifugation in Biochemistry. Academic Press, New York. SCHEIDEG~ER J. J. (1955) Une micro-m~thode de l'immuno6lectrophor~se. Int. Arch. Allergy appl. Immunol. 7, 103-110. SHIRASAWA K. (1965) T r a n s f e r r i n - - I I . Minor component of human transferrin. Proc. Japan Acad. 40, 610-615. SILBERZAHN P., RICHARD G. B. & CREYSSEL R. (1967) Isolation and study of proteins with hemopexic properties and of transferrin from carp (Cyprinus carpio) serum. Bull. Soc. Chim. Biol. 49, 495-506. SJOQUIST J. (1957) Determination of amino acids as phenyl thiohydantoin derivatives. Arkiv. Kemi 11, 129-135. SMALL P. A., JR. & LAMM M. E. (1966) Polypeptide chain structure of rabbit immunoglobulins--I. G-immunoglobulin. Biochemistry 5, 259-267. SMALL P. A., JR. & RESNICK R. A. A Fortran Program (PAS OOIC)for the Yphantis Meniscus Depletion Method of Molecular Weight Determination. Spinco Division, Beckman Instruments, Palo Alto, California. SMITHIES O. & HILLER O. (1959) T h e genetic control of transferrins in humans. Biochem.J. 72, 121-126. SPACKMAN D. H. (1962) Model 120B Amino Acid Analyzer. Beckman Instruments, Palo Alto, California. SUTTON H. E. • BOWMAN B. H. (1961) Genetic and chemical aspects of the transferrins. Proc. 2nd int. Cong. Human Genet., pp. 712-718. Istituto G. Mendel, Rome. THOMAS M. L. H. & MCCRIMMON H. R. (1964) Variability in paper electrophoresis patterns of the serum of land-locked sea lamprey, Petromyzon marinus Linnaeus. J. Fish Res. Bd Can. 21,239-246.
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TOMBS M. P., SOUTER F. & MACLAGAN N. F. (1959) The spectrophotometric determination of protein at 210 m/z Biochem ft. 73, 167-171. U T H E J . F. • TStlYUKI H. (1967) Comparative zone electropherograms of muscle myogens and blood proteins of adult and ammocoete lamprey, ft. Fish. res. Bd Can. 24, 1269-1273. WARNER R. C. & WEBER I. (1953) The metal combining properties of conalbumin, ft. Am. Chem. Soc. 75, 5094-5101. WARREN J. L. (1959) T h e thiobarbituric acid assay of sailic acids. J. biol. Chem. 234, 19711975. WILLIAMS J. (1962) A comparison of conalbumin and transferrin in the domestic fowl. Biochem.ff. 83, 355-364. WINDLE J. J., WIERSEMAA. L., CLARKJ. R. & FEENEY R. E. (1963) Investigation of the iron and copper complexes of avian conalbumin and human transferrins by electron paramagnetic resonance. Biochemistry 2, 1341-1345. WOODS K. R. & WANG K. T. (1967) Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. biophys. Acta 133, 369-370. YPHANTIS D. A. (1964) Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3, 297-317.
Key Word Index--Transferrins; polymorphic transferrin; Petromyzon marinus; sea lamprey; protein analysis; immunoautoradiography.
I S O L A T I O N AND PARTIAL C H A R A C T E R I Z A T I O N OF T R A N S F E R R I N IN T H E SEA LAMPREY, PETROMYZON MARINUS* R. O. W E B S T E R
a n d B. P O L L A R A
Departments of Pediatrics and Biochemistry, University of Minnesota Variety Club Heart Hospital, Minneapolis, Minnesota 55455, U.S.A. (Received 3 ffanuary 1969)
A b s t r a c t - - 1 . Transferrin was isolated and purified from sea lamprey serum by D E A E column chromatography in 0"005 M PO4 buffer, p H 8"0. 2. Lamprey transferrin has a molecular weight of 73,200-78,000 and a S20,w value of 5-5. 3. Six polymorphic forms of lamprey transferrin can be demonstrated. 4. Total amino acid analysis shows the absence of any appreciable amounts of arginine. 5. No free N-terminal amino acid was found. 6. Immunoradioautography shows the presence of two antigenically distinct iron-binding proteins.
INTRODUCTION THE TRANSFERRINSfrom blood serum have been variously called the fll metalbinding protein, siderophilin and transferrin. With ionic iron they form stable, salmon-pink complexes. In all serum transferrins examined thus far, elaborate genetic polymorphism has been demonstrated. All animals except man show a regular frequency of the different polymorphs. In humans, only one variant, type C, has appeared in high frequency in all populations (Feeney & Komatsu, 1966). The allelic forms are restricted to certain races and others only to certain kinships. No clear-cut physiologicaldifferences have been demonstrated between the various polymorphic types of transferrin. Multiple transferrins have been found by Dessauer et al. (1962) in over 150 representatives of amphibians and reptiles. Moeller& Naevdal (1966) and Creysse] et al. (1966) have found transferrin polymorphism in several different species of fish. The literature contains several other reports of polymorphism in all other classes of vertebrates (Blumberg, 1960; Arfors & Beckman, 1962; Beckman, 1962; Williams, 1962). The primary role of transferrin is that of iron transport (Hahn et al., 1942; Granick, 1946; Fletcher & Huehns, 1968). Titration and electrophoretic data (Warner & Weber, 1953; Malstrom et al., 1963; Windle et al., 1963) suggest that the binding site of iron in transferrin is made up of three phenolic groups plus bicarbonate. Two other nitrogen ligands have been implicated, either arginine, lysine or histidine or even a mixture of two of them. But, in general, the 509
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R . O . WEBSTER AND B. POLLARA
specific metal-binding properties of the transferrins m u s t depend upon the fundamental properties of the protein as well as u p o n the positioning and reactivity of the particular ligands (Bullen et al., 1967). Cyclostomes are the most primitive living vertebrates. T o g e t h e r with the extinct Ostracoderms they f o r m the super-class Agnatha which has followed an independent evolution since earliest vertebrate origins ( R u m e n & Love, 1963). T h e only living representatives today are the hagfish and lamprey. T h e sea lamprey has been shown to have iron-binding proteins which had in the past been confused with g a m m a immunoglobulins. Boffa et al. (1967a) have called attention to the lamprey transferrins and have shown this protein to be separate f r o m the i m m u n o globulin. O t h e r studies (Finstad & Good, 1964; Papermaster et al., 1964) on the immunological capacity of the lamprey demonstrated that this animal could produce antibody responses but only to a limited n u m b e r of antigens. T h e sea lamprey appears morphologically, anatomically and biochemically to be a connecting link between the lives of invertebrates and vertebrates (Applegate, 1950; L e n h e r t et al., 1956). Because the transferrins represent a group of serum proteins with a specifically defined function, i.e. iron binding and transport, it seemed important to characterize transferrin in a primitive vertebrate like the sea lamprey with the ultimate goal of determining some pattern of evolution of the properties and structure of this important protein.
MATERIALS AND M E T H O D S A.
Experimental animals Parasitic adults and non-feeding, sexually mature adults were used in the study. All fish were maintained at the United States Bureau of Commercial Fisheries Biological Laboratory, Hammond Bay, Michigan. The parasitic adults were kept in aquaria with aerated lake water and maintained on host fish ad lib. During the spring upstream spawning migration, sexually mature adults were trapped in live boxes in the Ocqueoc River, Hammond Bay, Michigan. The adults were held in large galvanized tanks and concrete raceways with circulating lake water. Mature adults ranged in size from 30 to 60 cm with an average length of 42 cm and average weight of 200 g. B.
Collection of whole lamprey serum Individual animals were bled by cardiac puncture after anesthetizing the fish with 4-styryl pyridine. The serum was kept in the cold at 4°C or frozen at - 2 0 ° C until used. C.
Preparation of antiserum Antiserum to whole lamprey serum was prepared in both rabbits and goats. Fresh lamprey serum (0'2 ml) was mixed with an equal volume of complete Freund's adjuvant and injected subcutaneously into adult New Zealand rabbits. The rabbits were injected once a week for the first month and then once every 2 weeks. Blood was drawn from the ear vein and serum collected after centrifugation of the coagulated blood specimen. Goats were injected once a week for 2 months with 0.2 ml of whole lamprey serum. Blood was obtained via the jugular vein.
ISOLATION AND CHARACTERIZATIONOF TRANSFERRININ SEA LAMPREY
D.
511
Isolation of lamprey transferrin by column chromatography
Transferrin from the lamprey was isolated by two different methods on diethylaminoethyl (DEAE) cellulose. I n Method 1 20-25 ml of pooled, normal serum from sexually mature lampreys was dialyzed against the starting buffer (0"006 M phosphate, pH 8"0) for 16-24 hr in Visking tubing (8 ram) with constant stirring in the cold. A column (2.5 x 50 cm) was packed with 180 ml of DEAE-cellulose equilibrated with the starting buffer. T h e column was eluted in a stepwise fashion utilizing the following discontinuous gradient of phosphate buffers: 0"006 M, 0"05 M, 0"075 M, 0"1 M and 0"2 M. All buffers were at p H 8"0. Three-ml effluent fractions were monitored at 280 m/z on a Beckman D U spectrophotometer. T h e absorption peaks were pooled and concentrated by means of positivepressure pervaporation. The concentrated pools were then dialyzed against the initial buffer to remove salts. Method 2 incorporated the use of a continuous gradient as outlined by Peterson & Sober (1959) modified by Bridges (personal communication). Serum from individual fish was labeled with iron-59. The labeled serum was placed on a small column (2.2 x 25 cm) after dialysis in the cold with constant stirring for 16-24 hr against the starting buffer to remove u n b o u n d iron-59. Fifty ml of each of the following buffers was used in each chamber: 1. 0"005 M phosphate, pH 8"0 2. 0"03 M phosphate, pH 7"88, 0"004 M NaC1 3. 0'06 M phosphate, p H 7"74, 0'003 M NaCI 4. 0"06 M phosphate, pH 7'59, 0-032 M NaCI 5. 0"06 M phosphate, p H 7.44, 0"061 M NaC1 6. 0'06 M phosphate, pH 7"29, 0"090 M NaC1 7. 0"06 M phosphate, pH 7"15, 0'119 M NaC1 8. 0"06 M phosphate, p H 7"00, 0"150 M NaC1 9. 0'06 M phosphate, pH 6"00, 0-300 M NaC1 T h e fractions were monitored at 280 m/z on the Beckman D U and were pooled and concentrated by pervaporation and later dialyzed against the starting buffer.
E.
Radioisotope labelling of serum
Fifteen/zc of iron-59 citrate solution were added per ml of serum or isolated transferrin fraction. The mixture was dialyzed in Visking cellulose tubing (8 ram) against multiple changes of 0"005 M phosphate buffer, pH 8"0. The dialysis was carried out for 16-20 hr in the cold with constant stirring to remove excess iron atoms.
F.
Microimmunoelectrophoresis
Immunoelectrophoresis was carried out as described by Scheidegger (1955) modified by Bridges (personal communication) using a gel containing 1"5 ~o (w/v) agar.
G.
Protein analysis
Relative protein concentrations were determined by measurement of the absorbance at 280 rn/z. More accurate protein concentrations were determined by the method of Folin & Ciocalteu (1927) as modified by Lowry et al. (1951). An alternative method for the spectrophotometric determination of protein concentration was the method of T o m b s et al. (1959).
H.
Vltracentrifugation analysis 1. Sedimentation velocity. Sedimentation velocity studies were carried out in the Spinco
Model E analytical ultracentrifuge. A 12-rnm wedge window, single-sector aluminum cell was used at a rotor speed of 59,780 rev/min. The temperature was maintained constant at 20°C by means of a R I T C unit. Schlieren optics were used with a red filter (655 m/z) and
512
R . O . WEBSTERAND B. POLLARA
Kodak spectroscopic plates (Type I-N). Sedimentation coefficients were calculated from the equations given by Schachman (1959). The S~0,w values at zero concentration was obtained by graphical extrapolation of the calculated S2o,w values. 2. Molecular weight determination (by ultracentrifugation or column chromatography). The molecular weight of transferrin was determined in the analytical ultracentrifuge at 20°C by the high-speed sedimentation equilibrium method using interference optics described by Yphantis (1964). Sapphire windows were used at all speeds and protein concentrations between 0'8 and 3.6 mg/ml were employed. A green filter (546 rn/~) was used. Kodak spectroscopic glass plates (Type II-G) were measured in a Nikon comparator after which the weight-average molecular weight was calculated by the least-squares fit of a In C vs. r ~ plot. The Z-average molecular weight was calculated from the equation of Yphantis (1964). Calculations were made with the aid of a Control Data Corporation 3300 computer using the Fortran program of Small & Resnick. The molecular weights of several transferrin samples were estimated by the column chromatography method of Andrews (1964) and Leach & O'Shea (1965) modified by Bridges (personal communication). An upward-flowing, temperature-controlled column (3"2 × 80 cm) was filled with G-200 Sephadex. One- to 2-ml samples were placed on the column at regular intervals so that no overlapping of protein peaks would occur. All of the runs were made at 4°C. Cytochrome-c, albumin and glutamate dehydrogenase were used as calibration standards. The column effluent was monitored at 210 m/z to establish the position of the protein peaks. The column volumes were estimated by pooling the collected fractions.
I.
N-terminal amino acid analysis Two methods were used for N-terminal amino acid analysis. The dansyl technique was used because of the small quantities required (m/zmoles) while the Edman method was used because of the ease of preparation and identification of the N-terminal amino acid derivatives. 1. The dansyl method. The dansyl reagent [1-dimethylaminonaphthalene-5-sulfonyl chloride (DNS-CI)] was used as suggested by Gray & Hartley (1963). Standard D N S amino acids were purchased from Gallard-Schlesinger (N.Y.). Chromatographic identification was achieved by the method of Woods & Wang (1967). 2. The modified Edman method. The Edman method (1953, 1960), as modified by Sjoquist (1957) and Blomback et al. (1966), was initially used to try to determine a short sequence of N-terminal amino acids. In each case 5/~moles of protein were used. The P T H amino acids were chromatographed using the method of Jeppsson & Sjoquist (1957). P T H amino acid standards were purchased from the Mann Research Laboratory, Inc., N.Y. Eastman chromagram sheets type K301R with fluorescent indicator were activated at 105°C 30 rain before use. Each side of a sheet in the direction of a chromatography was scrapped 1 cm wide in order to prevent front-line convection. The samples were applied 1"5 cm apart under a stream of nitrogen. J.
Amino acid analysis Total amino acid analyses were performed according to the method of Moore et al. (1958) and Spackman (1962) in a Spinco automatic amino acid analyzer Model 120B. The acid hydrolysis was carried out in 6 N HC1. The results were obtained in/zmole per cent, and converted to residues per mole of transferrin with the following equation : /zmoles amino acid X M.W. transferrin (rag) residues 1 mg transferrin 1 m-mole transferrin mole K.
Sialic acid assay The sialic acid content was determined by the thiobarbituric acid method described by Warren (1959).
ISOLATION AND CHARACTERIZATION OF TRANSFERRIN I N SEA LAMPREY
513
L. Polyacrylamide disc-electrophoresis Transferrin and other proteins were qualitatively separated by disc-electrophoresis involving the use of 7% polyacrylarnide gel as described by Ornstein (1964) and Davis (1964) modified by Hong (personal communication).
M. lmmunoradioautographs Radioautographs of the dipping type were done according to the method of Messier & Leblond (1957). Immunoelectrophoresis of the desired samples was carried out as previously described. Fifteen /zc of iron-59 were added to 0"2 ml whole lamprey serum and the mixture dialyzed against 0"005 M phosphate, pH 8"0, to remove unbound iron-59. The slides were developed for 7-14 days. RESULTS
A.
Isolation of pure lamprey transferrin
Stepwise elution of the DEAE-cellulose column with five buffers of increasing ionic strength (0.006-0.2 M phosphate, p H 8.0) resulted in five protein peaks of which the first peak contained the isolated transferrin. Careful attainment of column equilibrium with the starting buffer resulted in isolation of pure transferrin. Purity was checked by immunoelectrophoresis and by analytical ultracentrifugation. A representative chromatogram of whole lamprey serum separation can be seen in Fig. 1. DEAE
WHOLE LAMPREY SERUM
200
I
I. 6 0
OD2Bo 1.20
8
I
30
7O
I10
150
Fraction No.
FIG. 1. Chromatogram of pooled whole lamprey serum on DEAE-cellulose using step-wise elution. All buffers at pH 8'0. Separation of individual lamprey serum labeled with iron-59 was effected by the use of a small DEAE-cellulose column and a continuous gradient for elution.
514
R. O. WEBSTrRAND B. POLLARA
The gradient was not (transferrin) was eluted spontaneous release of serum separation using DEAE
applied to the column until after the first protein peak from the column. Use of a continuous gradient minimized radioactivity. A typical chromatogram of whole lamprey a continuous gradient is shown in Fig. 2.
WHOLE
LAMPREY
SERUM CONTINUOUS GRADIENT
1.40
7O
1.20
6O
1.00
5O
.80
4O
CPM
X[O 2
ODzeo o--.,o
.60
50
401
2O
]0
__
20
25
60
70
1 0
l
80 Frection
i I00
IIO
No.
Fic. 2. Chromatogram on DEAE-cellulose of whole lamprey serum using a continuous gradient of 0"005 M PO4, pH 8"0-0"06 M PO4 with 0'300 M NaCI. Those samples of transferrin containing iron-59 were placed on an Andrew's column containing G-200 Sephadex in order to estimate the molecular weight. Figure 3 shows a typical chromatogram in which the radioactive peak corresponds directly to the protein peak of transferrin. Analysis of the isolated transferrin samples by microimmunoelectrophoresis showed that the lamprey transferrin migrates to the cathode as Boffa et aL (1967a) had previously described. B.
Protein analysis
The spectrophotometric method of Tombs et al. (1959) proved to be the method of choice in determining the protein concentration of both lamprey transferrin and serum. The most noticeable fact in the measurement of total serum proteins in the lamprey is the large decrease in protein concentration as the animal matures. Quantitative experiments showed that the total serum protein level varied from 74.0 to 34.6 mg/ml in the parasitic adults and from 70-2 to 21.1 mg/ ml in the sexually mature adults. This represents a possible decrease of as much
ISOLATION AND CHARACTERIZATION OF TRANSFERRIN IN SRA LAMPREY
515
as 71.4 per cent. T h e highest concentration of pure serum transferrin isolated from one individual fish was 4.95 mg or 9"3 per cent of the total serum protein. Tronsferrin - Fe 59 on Sephodex G - 2 0 0 O.06M PO4 ~" 0.075M KCI
lO0
.800
8O !
ODzto .6oo ¢.-,-..o
60
.400
40
200
20
260
I
I
T
270
280
290
CPM XlO z
Froction No.
FIC. 3. Chromatogram of transferrin labeled with iron-59 on G-200 Sephadex (0"06 M PO4 with 0"075 M KC1).
C.
Ultracentrifugation studies 1. Sedimentation velocity. Sedimentation velocity analyses of transferrin in
varying concentrations were carried out in 0.005 M phosphate buffer, pH 8.0. Sedimentation values and concentration are listed in Table 1. Concentrations of transferrin were plotted against sedimentation values corrected to standard conditions (Fig. 4). At infinite dilution the S~o,w value was 5.5. TABLE l m S U M M A R Y OF Sso,t o VALUES FOR TRANSFERRIN (DETERMINED AT ROTOR SPEED OF
59,780 rev/min) Cone. (mg/ml)
S~o,w
1.12 1.71 4.05 6.11 7-50
5.42 5.37 5-25 5.09 5-01
2. Molecular weight determination. Equilibrium molecular weights of transferrin were obtained with the multichannel cell of Yphantis for three different concentrations of transferrin at two speeds. T h e runs were made in phosphate
516
R. O. WEBSTER AND B. POLLARA TRANSFERRIN
SPEED
59.780
S20,W
501 I
I
i L__
I_
I0
I
2.0
1
30
I ___i
40
50
I
6.0
I
ZO
8.0
Oonc. mg/ml
Fro. 4. Relation of S2o,w to concentration of transferrin.
-251
I i
-2 7 !
ii -5. r ~ I
J -351
I
InO
-3.9~ - 4 3~~ ' ~ * / I i
-47[ i I[____J___ 600
42550
.700
I
.800
__ I
900
.__~
43000
I
.~6o
£
.200
J
.300
L 4~00
.500
Radius Squared
Fzc. 5. Relationship of natural logarithim of concentration to radius squared showing homogeneity and ideal conditions.
ISOLATION AND CHARACTERIZATION OF TRANSFERRIN IN SEA LAMPREY
517
buffer (0-005 M, pH 8-0) at 20°C. The average molecular weight of the protein was 73,200 + 2200, assuming a partial specific volume, ~ of 0.73. A representative plot of In concentration vs. radius squared is shown in Fig. 5. Examination of the plot of the logarithim of the concentration vs. the radius squared yields evidence of the degree of homogeneity or nonideality. As "Small & Lamm (1966) point out the nonlinearity of the plot where the plot has a slight but definite upward curvature is an indication of heterogeneity, whereas a downward curvature is an indication of nonideal conditions. The determination shown in Fig. 5 approaches ideal conditions and homogeneity.
D.
Molecular weight determination by column chromatography
The molecular weight of transferrin was also determined by use of an Andrew's column in addition to the high-speed equilibrium method of Yphantis. The standards and their respective molecular weights used in the calibration of the column are as follows: cytochrome-c, 12,400; albumin, 67,000; and glutamate dehydrogenase, 250,000 (Andrews, 1964). The column volumes of the standards and of several different transferrin fractions are given in Table 2. The standard curve from which the molecular weight of transferrin was obtained is shown in Fig. 6. The molecular weight of transferrin by this method was estimated as 78,000. TABLE 2--CoLuMN
Average
VOLUMES OF K N O W N STANDARDS AND LAMPREY TRANSFERRIN RUN ON A N D R E W ' S COLUMN (VALUES I N ml)
Cytochrome-c
Albumin
Glutamate dehydrogenase
525 540 528 535 519 523 543
338 356 343 345 330 334 346
229 236 226 225 222 222 223
530
342
226
Transferrin
Estimated weight
336 337 337 334
79,000 78,000 78,000 80,000
336
78,750
Average
E. Amino acid composition The amino acid composition of lamprey transferrin is given in Table 3. Tryptophan and cysteine, destroyed by acid hydrolysis, were not measured due to the
518
R. O. WEBSTERAND B. POLLARA
large amounts of protein needed for reaction. Since the amide forms of the dicarboxylic amino acids were converted to the acid forms upon acid treatment, glutamic acid and glutamine were reported together, as were aspartie and asparagine. Only a slight trace of arginine was found to be present in the pooled sample of transferrin. STANDARD CURVE ANDREWS COLUMN I00 .80 60
40
Glulomo/e Oehydrogenose 20
MWxlO 4
I0
Tronsferr/n
8 6
Cyfochrome C I
I
1
L~
~
T
I00
200
~00
400
500
Effluent
Volume .(ml)
FIG. 6. Standard elution curve from Andrew's column (G-200 Sephadex, 0"06 M PO4 with 0"075 M KC1, pH 8"0).
F.
Sialic acid assay
Three different samples of isolated transferrin were subjected to the sialic acid assay of Warren (1959). Standards made up of pure N-acetyl neuraminic acid were run at the same time as the isolated transferrin samples. In every case, there were no residues of sialic acid detected.
G.
N-terminal amino acid analysis and sequence analysis Experiments to determine the N-terminal amino acid of transferrin were done by two methods. Using the Edman method, the first or N-terminal residue did not migrate with either solvent system used, suggesting that the N-terminal was
ISOLATIONAND CHARACTERIZATIONOF TRANSFERRININ SEA LAMPREY
519
cysteic acid. An attempt was m a d e to couple and cleave off the second amino acid residue, T h i s yielded a similar spot. TABLE 3 - - A M I N O ACID COMPOSITION OF LAMPREY TRANSFERRIN*
Trial 1
Asp Thr Ser Glut Prol Gly Ala Val Met Ile Leu Tyr Phe Lys His Arg
Trial 2
Trial 3
Res/
Res/
Res/
Res/
Res/
Res/
73,200 g
10,000 g
73,200 g
10,000 g
73,200 g
10,000 g
38.4 22"2 32"0 29"2 29'2 22"6 61"8 39"2 12"8 23"2 39"6 12'0 25"4 49.8 6.6 Trace
5.2 3"0 4"4 4"0 4"0 3"0 8"4 5'4 1"8 3'2 5"4 1"6 3"4 6.8 1.0
57.2 35"8 49-8 42"0 29"0 36"8 81"2 49"8 15"8 22"2 40"8 18"8 28"2 --~ . .
7.8 5'0 6"8 5"8 4"0 5"0 11-2 6"8 2'2 3'0 5"6 2"6 3"8 --
39.8 24"0 34"2 29-2 18'4 24"8 55"8 33"0 9"8 15'8 27"6 10"4 20"8 --
5.4 3"2 4"8 4'0 2"6 3'4 7"6 4-6 1"4 2"2 3"8 1'4 2"8 --
. .
. .
. .
* Determination did not account for tryptophan, cysteine, iron content nor carbohydrate content. Not determined. Since b o t h of these derivatives behaved in this manner, it was thought that the spot could represent degradation products. Serine and threonine, usually destroyed by acidic conditions, were checked for b y using the alternative E d m a n m e t h o d (Guidotti et al., 1962). No P T H residues were found u p o n c h r o m a t o g r a p h y of the material obtained f r o m the reaction mixture indicating that the N - t e r m i n u s might be blocked. Since an N - t e r m i n a l group could not be detected using the E d m a n method, the highly sensitive dansyl m e t h o d was employed. I t was impossible to obtain fluorescent spots f r o m the transferrin reaction mixture corresponding to the labelled amino acid standards suggesting that the N - t e r m i n u s was indeed blocked (possibly by an aeetyl group or pyrrolidone carboxylic acid).
H.
Polyacrylamide disc-electrophoresis
U p o n examining isolated and purified transferrin fractions by polyacrylamide gel discs it was seen that as m a n y as six different transferrin bands were found in a pooled sample (Fig. 7). T h i s suggested the presence of six differently charged transferrin species. W h e n the gels were sliced horizontally and counted in a g a m m a
520
R . O . WEBSTER AND B. POLLARA
counter, radioactive peaks obtained coincided exactly with the protein bands revealed by staining. Thus, specific iron-binding capacity was present in all six bands. Transferrin fractions obtained from individual animals and labeled with iron-59 were also placed on microdisc-electrophoresis where only one band was found. I.
Immunoradioautographs The radioautographs of the isolated transferrin samples from sexually mature adults showed a single line of gamma mobility. On the other hand, those immunoradioautographs done on whole lamprey serum showed two different patterns. Whole serum from sexually mature adults (Fig. 8) showed the same single band as the sample of isolated and purified transferrin. However, whole serum from the parasitic type adults (Fig. 9) contained two iron-binding proteins. These two iron-binding proteins were antigenically distinct as the protein arcs crossed each other. There was a homogeneous, fast migrating band and one very heterogenous, slow migrating band.
DISCUSSION In several reports (Boffa et al., 1966, 1967a, b), it has been suggested that lamprey transferrin migrates in the area of gamma immunoglobulins as shown on immunoelectrophoresis. Subsequently, it has been seen (Pollara, unpublished observation) that the immunoglobulins in this animal have alpha or fast beta mobility as shown on microimmunoelectrophoresis. It has also been demonstrated, by use of the functional properties of transferrin binding iron-59 and immunoglobulin precipitating Brucella antigen, that these proteins possess distinctly different electrophoretic mobilities. A.
The nature of transferrin in the sea lamprey In many respects lamprey transferrin does not seem to differ widely from the other characterized transferrins. The molecular weight of lamprey transferrin (73,200-78,000) is like the transferrins of carp (70,000) (Silberzahn et al., 1967) and human (73,000-78,000) (Roberts et al., 1966). There is a difference in the value obtained by ultracentrifuge method (73,200) and the value obtained by the Andrew's columns (78,000). A partial specific volume (17) was assumed to be 0.73, based on reported values for other transferrins (Putnam, 1965). This assumption may account for the discrepancy in the molecular weights. The corrected sedimentation velocity value of 5"5 is slightly higher than the value given by Boffa et al., (1967a, b) of 5.1. Roberts et al. (1966) described the dependence of sedimentation velocity of human transferrin on pH. Boffa's data did not include conditions which were used to perform the ultracentrifuge experiments. Hence, no comparison can be made between the different sets of data. The dependence of the sedimentation velocity of lamprey transferrin on pH was not investigated in this study.
+ FIG. 7. Disc-electrophoresis of isolated transferrin obtained from pooled serum (anode at bottom).
FIC. 8. Immunoradioautographs of sexually mature lamprey serum tagged with iron-59 (15 /zc/rnl of serum). Whole lamprey serum in antigen wells with goat anti-whole lamprey serum in antibody trough.
L
FIG. 9. Immunoradioautographs of whole parasitic lamprey serum tagged with iron-59 (15/zc/ml of serum). Lamprey serum in antigen wells with goat anti-whole lamprey serum in antibody trough.
ISOLAT~ONANDCHARAC~mZATIONOr ~ANSFrRmNm SEALA~P~
521
The absence of any appreciable amount of arginine in the total amino acid analysis provides considerable evidence that this amino acid is excluded from consideration as one of the possible N-ligands of the binding site of iron in transferrin. Buttkus et al. (1965) have concluded from blocking studies that "if two nitrogen ligands are directly involved in metal binding, these nitrogens are probably not contributed to by the e-amino groups of lysine". This would leave only histidine as the likely amino acid in the role of N-ligand in lamprey transferrin. This would be entirely comparable to the situation as it is found in the binding site of oxygen in hemoglobin. N-terminal amino acid analysis failed to reveal an active N-terminal amino acid. Based on an estimated molecular weight of 70,000, an adequate amount of protein was used in these determinations. Absence of a reactive amino acid suggests that the N-terminus may be blocked by an acetyl group or pyrrolidone carboxylic acid. The presence of these groups as N-terminals has been seen in a number of other proteins (Press et al., 1966; Putnam et al., 1967). The reduction of the serum protein level As the lamprey matures beyond the parasitic stage, a marked decrease occurs in the serum proteins. Uthe & Tsuyuki (1967), using polyacrylamide disc-electrophoresis of blood proteins of adult and ammocete lampreys, showed a significant decrease in the total amount of protein. Rall et aL (1961) showed that the alpha globulins decrease from 70 per cent in the larval forms to 37 per cent in the mature adult. In our quantitative protein study, the total serum protein levels were seen to decrease over 70 per cent as the lamprey matures from the parasitic stage to the sexually mature stage. As Thomas & McCrimmon (1964) point out, the parasitic lamprey represents a rapid period of growth in the life history of the sea lamprey in the open lake which lasts for 12-18 months. During the spring migration which follows the parasitic feeding stage, lamprey ascend tributary streams to spawn. The lamprey do not feed during the spawning migration and die following the spawning process. Applegate (1950) has demonstrated morphologically that a severe involution of the digestive tract takes place once feeding has stopped. Thus, it seems that the serum proteins are used as metabolites and basic building blocks as the body requires them. Since there is no other means of obtaining any foodstuffs, the serum could be a primary protein source resulting in a decreasing protein concentration as the animal ages. B.
C.
Polymorphism in lamprey transferrin The elaborate polymorphism of transferrin found in many other vertebrate animals is also found in the sea lamprey. Boffa et al. (1967a) reported finding five transferrin bands in the lamprey by the method of radioautography after electrophoresis on starch-gel blocks and paper strips. Since several authors (Ornstein, 1964; Rausch et al., 1965; Margolis & Kenrick, 1967) mentioned that fine protein 'separations are posisble on polyacrylamide gel, it was elected to use this method using transferrin labeled with iron-59. These studies showed the existence of six
522
R . O . WEBSTER AND B. POLLARA
different transferrin bands. When the gels were sliced into equal parts and counted in a radiation counter, the radioactive peaks corresponded directly to the six stained protein bands. Parker & Beam (1961a, b) found that starch-gel electrophoresis of labeled human transferrin C - - C mixed with neuraminidase from Vibrio cholerae revealed a step-wide pattern of five bands whose intensities varied with neuraminidase concentration. They attributed the different migrating species to the amount of sialic acid attached to the transferrin. The fastest moving band coincided with untreated transferrin and contained approximately four sialic acid residues per molecule o5 protein. On the other hand, the slowest moving band represented complete removal of sialic acid. Subsequent analysis of other transferrin variants with different migration rates showed that they all contained four sialic acid residues per molecule of transferrin. So it became apparent that the difference in migration was not due to the protein having less than four sialic acid residues. The absence of sialic acid residue in lamprey transferrin indicates that the observed differential rates of migration would have to be due to the presence of different electric charges on the protein. This can be accounted for by substitution of different amino acids within the protein. Such substitutions could be controlled by alterations of the genome directing transferring synthesis. The results suggest a hypothesis; that the genetic polymorphism shown here may be controlled by three alleles, A, B and C. The six variants could be made up of the following genotypes: AA, BB, CC, AB, AC and BC. In a review article, Giblett (1962) mentioned that Smithies & Hiller (1959) suggested the symbol Tffor the autosomal locus with the superscript letters for the type of protein variant. Giblett further states that the fact that no human serum has been conclusively shown to contain three transferrins provides some indirect evidence that the transferrin variants represent a series of alternative dominant alleles at a single locus. Such also could be the case in the sea lamprey. It is doubtful that the variants are due to varying degrees of aggregation of polymer types of structure. If such were the case, it would have been detected in the Yphantis equilibrium run. There would also have been more than one peak observed while doing the sedimentation velocity experiments. Furthermore, the high order of sensitivity using iron-59 labeled transferrin on G-200 Sephadex is good evidence favouring monomer over polymer types of structure.
D.
Two iron-binding proteins in the sea lamprey One of the least expected discoveries in this study was the presence of two antigenically distinct iron-binding proteins in the lamprey. To further complicate the picture, the two iron-binding proteins were found only in the parasitic animals and not in the sexually mature animals. The immunoradioautographs show a complete immunological non-identity between the two proteins. The slower migrating, heterogeneous band eluted as the first peak on DEAE chromatography. This was identified as the lamprey transferrin. The fast, migrating homogeneous peak eluted with the last half of the fourth protein peak. Only the transferrin was
I S O L A T I O N AND CHARACTERIZATION OF TRANSFERRIN I N SEA LAMPREY
523
chromatographically pure as shown by microimmunoelectrophoresis. The second iron-binding protein was contaminated with several of the alpha-migrating proteins. No attempt was made to isolate this protein due to the extremely small amounts of protein available. The presence of two types of iron-binding proteins in other animals has been mentioned only briefly. Reports have been made involving rats (Gordon, 1963; Gordon & Louis, 1963), hagfish (Manwell, 1963) and man (Shirasawa, 1965). Only in the latter case has any investigation been done. Shirasawa separated these two components of transferrin on TEAE-cellulose chromatography but did not isolate the second component. He estimated that the minor component of transferrin C - - C comprised about 5-10 per cent of the radioactivity of the major component. The radioactivity of the second lamprey iron-binding protein was about 35 per cent of the transferrin peak, although this may merely reflect differences in iron-binding capacity. Lai & Kirk (1960) and Sutton & Bowman (1961) have reported a monkey with three iron-binding proteins. It has not been determined yet whether this phenomenon is due to a single gene determining more than one protein band or the presence in some monkeys of more than one transferrin locus. The disappearance of the fast migrating, homogeneous band in the sexually mature animal could be accounted for in two ways. The first possibility is that the fast migrating form is an early precursor form of transferrin which undergoes a transformation to one of the six allelic variants of the slower migrating form of transferrin. The other possibility is that upon the involution of the lamprey's digestive system, this protein is expendable and is used for nutritional purposes. SUMMARY Lamprey transferrin was isolated from whole serum by means of DEAE column chromatography. The isolated transferrin has been studied by means of immunological, physical and chemical methods. The purification of lamprey transferrin was accomplished by the elution of protein from a DEAE-cellulose column with the starting buffer (0.005 M phosphate, pH 8-0). Gel electrophoresis of the isolated transferrin fraction showed six electrophoretically different migrating bands. These polymorphs could be explained by the presence of three allelic autosomal genes of equal dominance. Quantitative analysis of the total serum proteins of the lamprey show a marked decrease as the lamprey ages from the parasitic to the sexually mature stage. This decrease ranges up to 70 per cent of the peak found in the parasitic fish. Upon reaching sexual maturity, the digestive tract of the lamprey involutes so that no further feeding of the fish can take place. Hence, the serum proteins are thought to be used as metabolites in required body functions. Immunoradioautography of parasitic lamprey serum reacted with iron-59 shows two antigenically distinct iron-binding proteins. One is a fast, migrating, homogeneous band and the other a slower migrating, heterogeneous band. The slower migrating band is the one that was isolated on DEAE chromatography and characterized. On sexual maturation, the fast migrating iron-binding protein
524
R. O. WEBSTERAND B. POLLARA
disappears f r o m the serum suggesting a possible functional difference in these proteins. Ultracentrifuge studies show a S~0, w value of 5.5 and a molecular weight of 73,200 + 2200. Estimation of the molecular weight on G-200 Sephadex chromatography gave an average value of 78,000. T h e absence of any sialic acid residues was confirmed. Reaction with the E d m a n and dansyl reagents failed to demonstrate a free N - t e r m i n a l amino acid suggesting that the N - t e r m i n a l residue is blocked. Total amino acid analysis shows the absence of arginine, thus excluding this amino acid f r o m participating in the binding site of iron and indicating histidine as the probable nitrogen ligand in the binding site. Acknowledgements--We would like to thank Dr. Robert A. Bridges for many helpful discussions and suggestions, and Mr. John H. Howell for his co-operation in maintaining the lamprey at Hammond Bay. Aided by grants from the National Foundation and U.S. Public Health Service (AI-08372).
REFERENCES ANDREWS P. (1964) Estimation of the molecular weights of proteins by Sephadex gelfiltration. Biochem. ff. 91, 222-233. APPLEGATE V. C. (1950) Natural history of the sea lamprey. U.S. Dept. Interior, Special Sci. Rep. Fisheries 55, 1-237. ARFORS K. E. & BECKMANL. (1962) Phylogentical and immunological aspects of primate serum transferrins. Hereditas 48, 132-137. BECKMAN L. (1962) Slow and fast transferrin variants in the same pedigree. Nature, Lond. 194, 796-797. BLOMBACK B., BLOMBACK M., EDMAN P. •
HESSEL B. (1966) H u m a n fibrinopeptides,
isolation, characterization and structure. Biochim. biophys. Acta 115, 371-396. BLUMBERG B. S. (1960) Biochemical polymorphisms in animals: haptoglobins and transferrins. Proc. Soc. exp. Biol. Med. 104, 25-28. BOFFA G. A., FINE J. M., DRILHON A. 8¢ AMOUCH P. (1967a) Irnmunoglobutins and transferrin in marine lamprey sera. Nature, Lond. 214, 700-702. BOFFA G. A., FINE J. M. &; FAURE A. (1967b) NouveUes donn6es sur la transferrine et les immunoglobulines de la lamproie marine. Bull. Soc. Chim. Biol. 49, 433-445. BOFFA G. A., MARINELLIG., DBILHONA. & FINE J. M. (1966) Evidence for and isolation of transferrin in the marine lamprey. C.r. hebd. Sdanc. Acad. Sci., Paris 262, 2294-2297. BULLEN J. J., ROGERS H. J. ~% CUSHNIE G. H. (1967) Abolition of passive immunity to bacterial infections by iron. Nature, Lond. 214, 515-516. BUTTKUS H., CLARK J. R. & FEENEY R. E, (1965) Chemical modifications of amino groups of transferrins: ovotransferrin, human serum transferrin and human lactotransferrin. Biochemistry 4, 998-1005. CREYSSEL G., RICHARD G. B. & SILBERZAHNP. (1966) Transferrin variants in carp serum.
Nature, Lond. 212, 1362. DAVIS B. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Annls. N . Y . Acad. Sci. 121, 404-427. DESSAUER H. C., Fox W. & HARTWIG Q. L. (1962) Comparative study of transferrins of Amphibia and Reptilia using starch-gel electrophoresis and autoradiography. Comp. Biochem. Physiol. 5, 17-29.
ISOLATION AND CHARACTERIZATIONOF TRANSFERRININ SEA LAMPREY
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Key Word Index--Transferrins; polymorphic transferrin; Petromyzon marinus; sea lamprey; protein analysis; immunoautoradiography.