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Modification of carboxyl groups of transferrin
BIOCHIMICAET BIOPHYSICAACTA
37
BBA 35612 MODIFICATION OF CARBOXYL GROUPS OF T R A N S F E R R I N
ANATOLY BEZKOROVAINY AND DIETMAR GI{OHLICH Biochemistry Department, Rz~sh-Presbyterian-St. Luke's 3Iedical Center, Chicago, Ill., 60612, and Department of Biological Chemist~iv, University of Illinois College of Medicine, Chicago, Ill., 6o6z2 (U.S.A.)
(Received March I6th, I97o)
SUMMARY Carboxyl groups of transferrin were coupled with glycine methyl ester using a water-soluble carbodiimide. The iron-binding activity of the modified protein did not change until some 16 carboxyl groups had been modified. The activity then fell in proportion to the number of groups modified until no iron binding was seen with 62 carboxyl groups modified. Such a slow drop in activity was interpreted to indicate that the carboxyl groups do not directly participate in the iron-binding reaction and that the activity was lost because of a conformational change in the protein structure. Experiments involving competition of native transferrin and EDTA foi iron indicated that the iron-binding activity of the former declined with increasing EDTA concentrations in a gradual manner. Similar experiments with transferrin with 15-17 carboxyl groups blocked by the glycine methyl ester showed that half of the iron-binding activity was lost with very low EDTA concentrations, remained at the 50% level over an EDTA concentration range of 0.0025 o.oi M and then commenced to decline in a gradual manner. It was concluded that the more reactive carboxyl groups are situated in the vicinity of only one of the two iron-binding sites of transferrin and that their modification then destabilizes the coordination complex. Very few if any reactive carboxyl groups are situated in the vicinity of the other iron-binding site. This finding supports the idea that the environments of the two iron-binding sites of transferrin are not identical.
INTRODUCTION Transferrin, the iron-binding protein of human serum, is said to bind two atoms of iron for each protein molecule. Each iron-binding site has been assumed to consist of two histidyl residues, 2-3 tyrosyl residues, and one bicarbonate ion 1. Whereas the binding of iron by nitrogen atoms has been established by both electron spin resonance 1 and chemical modification 2 studies, the role of tyrosyl residues has been surmised largely through indirect means, such as spectrophotometric titration data a and Abbreviation: CMC, I-cyclohexyl-3(2-morpholino-4-ethyl) carbodiimide methyl p-toluene sulfonate. Biochim. Biophys. Acla, zl 4 (t97o) 37 43
38
A. BEZKOROVAINY, D. GROHLICH
chemical modification w o r k 2,4. I t is t h u s i m p o r t a n t to i n v e s t i g a t e the possibility of the p a r t i c i p a t i o n of other a m i n o acid side chains in the iron-binding reaction. One such v e r y likely possibility are t h e c a r b o x y l groups, which, as c o m p o n e n t s of o t h e r compounds, are k n o w n to complex w i t h iron to form coordination derivatives. The purpose of the present c o m m u n i c a t i o n is to i n v e s t i g a t e the role of c a r b o x y l groups in the b i n d i n g of iron b y transferrin and their p a r t i c i p a t i o n in the m a i n t e n a n c e of the t e r t i a r y s t r u c t u r e of this protein. MATERIALS AND METHODS Chemicals The transferrin used in this w o r k was p u r c h a s e d from Hoechst L a b o r a t o r i e s ( W o o d b u r y , N.Y.). I t s molecular weight was assumed to be 83 ooo. Glycine m e t h y l ester hydrochloride a n d I-cyclohexyl-3(2-morpholino-4-ethyl) c a r b o d i i m i d e m e t h y l p-toluene sulfonate (CMC) were p u r c h a s e d from K a n d K L a b o r a t o r i e s (Plainview, N.Y.). All other chemicals were of reagent grade p u r i t y a n d were o b t a i n e d from F i s h e r Scientific Co. (Chicago, Ill.). Methods The coupling of t l a n s f e r r i n with glycine m e t h y l ester was done at p H 4.75 at 25 ° in an a u t o m a t i c t i t r a t i o n device according to the m e t h o d of HOARE AND KOSHLAND5. The c o n c e n t r a t i o n of CMC was v a r i e d for the purpose of o b t a i n i n g various degrees of modification of transferrin. A t y p i c a l e x p e r i m e n t was done as follows: 1.68 g of glycine m e t h y l ester-HC1 was dissolved in 7.5 ml water. The p H was a d j u s t e d to 4.75 with 5 M N a O H , a n d lOO-15o m g transferrin were then dissolved in this solution. A t zero t i m e O.l-O.4 g solid CMC was added, the reaction m i x t u r e was m a i n t a i n e d at p H 4.75, and 2-ml aliquots were w i t h d r a w n at various t i m e intervals (2-24o rain) a n d p i p e t t e d into 5 ml of o.5 M a c e t a t e buffer at p H 5.o to s t o p the reaction. These were t h e n d i a l y z e d against w a t e r a n d lyophilized. Controls were done b y i n c u b a t i n g all c o m p o n e n t s as described above, b u t o m i t t i n g the CMC. I r o n - b i n d i n g analyses of transferrins were p e r f o r m e d as follows: 5 mg of the n a t i v e or modified transferrin was weighed into a 13 m m × IOO m m test tube. To the p r o t e i n was then a d d e d I ml of a solution containing excess iron (this solvent was p r e p a r e d b y weighing out a p o r t i o n of F e N H 4 ( S Q ) 2, a d d i n g a sufficient volume of O.I M N a H C O 3 to m a k e a f i n a l Fe 3+ concentration of 2.8 .lO 4 M, a n d then a d d i n g a q u a n t i t y of solid t r i - s o d i u m c i t r a t e to just bring the iron into solution), or to the 5 m g p r o t e i n was a d d e d 0.88 ml w a t e r followed b y o. 12 ml of I • lO -3 M Fe 3+ in o.I M N a H C O a {prepared as above). The l a t t e r procedure p r o v i d e d a stoichiometric a m o u n t of iron. The solutions were p e r m i t t e d to s t a n d for I h at room t e m p e r a t u r e a n d their absorbbances were then m e a s u r e d at 465 n m a n d 280 n m (I : IO dilution). The iron-binding c a p a c i t y of the transferrin samples was then expressed as a r a t i o of t h e a b s o r b a n c e at 465 nm and 280 n m (ref. 6). F u l l y active transferrin h a d an A~65nm/A2sonm r a t i o of o.o48 o.o5o, whereas fully i n a c t i v e transferrin was assumed to have the A46 a nm/ ./128o nm r a t i o at o. In some instances it was necessary to c o m p a r e the s t a b i l i t y of the iron-transferrin complex in the modified p r o t e i n a n d n a t i v e protein. This was accomplished b y perm i t t i n g the transferrin p r e p a r a t i o n s to c o m p e t e for iron with various q u a n t i t i e s of Biochim. Biophys. Acta, 214 (197° ) 37-43
MODIFICATION
OF TRANSFERRIN
3 ()
ethylenediamine tetraacetic acid (EDTA) at pH 7.4. The procedure used was a modification of that described by BOTHWELL et al.V: 5-mg portions of transferrin were weighed into 13 m m × IOO mm test tubes. To each tube was added I ml of o.oo25-o.o5 M EDTA adjusted to pH 7.4. This was then followed by o.12 ml of the I .IO -3 M l"e~÷ solution (see above). The reaction mixtures were then placed in a water bath at 37 ° for I h, after which the A46~ nm/A280 nm ratios were determined.
Analytical procedures The amount of glycine incorporated into transferrin was measured via the automatic amino acid analyzer. The Spinco Model I2oC instrument was used according to the procedure of MOORE et al. 8. Prior to the analysis, the transferrin sample was hydrolyzed with 5.7 M HC1 for 24 h at IiO ° in a sealed tube previously flushed with nitrogen. The sample was repeatedly lyophilized after the hydrolysis. The number of carboxyl groups modified was determined by subtracting the number of intrinsic glycine residues in transferrin (50 as given by SCHULTZEngI) HEREMANS9 for a particle with a molecular weight of 83 ooo) from the total number of glycine residues present in a given sample. The precision of the amino acid analyses was approx. 5 %All speetrophotometric measurements were done in a Zeiss PMQ II instrument using the I-ml semi-micro cells. Optical rotation measurements were done in a Perkin Elmer Model 141 apparatus at 25 ° and 589 nm using the i-ml cells (I dm). Ultracentrifugal analyses were done in a Spinco Model E apparatus at 2o ° at full speed. Moving-boundary eleetrophoresis was performed in an Antweiler mieroelectrophoresis apparatus at 60 V (1. 4 mA) in the 0.05 M veronal buffer at pH 8.6. Proteins were analyzed at concentrations of 20 mg/ml. RESULTS
1"he incorporation of glycine into tran@rrin Different amounts ofglycine could be incorporated into transferrin by (a) varying the concentration of CMC in the reaction mixtures and (b) varying the time of reaction. The results of such experiments are shown in Table I. It may be noted that the')lighest number of glycine residues incorporated into transferrin was 54 per protein molecule. TA BLE
I
INCORPORATION
Data
OF GLYCINE
are numbers
Time of reaction
INTO
TRANSFERRIN
of glycine residues
incorporated
per molecule
of transferrin
C M C conch. (g/±o ml reaction m i x t u r e ) ;
(rain) o.I 2 5 15 3° 6o 90 240
o.2
7 16
29 17
0.3 i~ 21 37 39 44 --
0.4
27 ----
54
13iochim. Biophys. qcta, 2 t 4 (~967) 3 7 - 4 3
40
A. BEZKOROVAINY, D. GROHLICH
Fig. i. Moving-boundary electrophoresis of native and modified transferrins. I n b o t h cases the p h o t o g r a p h s were taken 4 ° rain after commencing the run. A. Native transferrin. B. Transferrin with 3 ° carboxyl groups modified.
Modified and native transferrin samples were analyzed by the method of movingboundary electrophoresis. In general, the mobilities of modified transferrins were retarded in proportion to the number of glycine groups introduced. A representative electrophoresis pattern is shown in Fig. i.
0.05
O~ O -,
O.OSL~ A46,,~S 0.04 I~O~o A280 0,03
O~
0.04
A465
o
O
0,03 i
A280 0.02
~
O
~
o
IL0 20 30 40 50 60 TiME (min) ~ O
0.01 I
I
I
J
10
20
30
40
NUMBER OF GLYCINE R E S I D U E S I N T R O D U C E D T R A N S F E R R I N MOLECULE
50
60
I N T O EACH
Fig. 2. Iron-binding activity of transferrin as a function of the n u m b e r of carboxyl groups modified. The inset g r a p h presents the rate of transferrin inactivation in the presence of o.o 7 31 CMC
Biochim. Biophys. Acta, 214 (197 o) 37 43
MODIFICATION OF TRANSFERRIN TABLE
41
II
COMPETITION
OF
tDWa
Protein concentrations
E D T A concn. (M)
o 0.0025 0.0050 0.0075 O.OLOO 0.0250 0.0500
WITH
VARIOUS
TRANSFERRIN
PREPARATIONS
FOR
IRON
w e r e 5 m g / m l in all c a s e s .
A46~nrn/A~ s o
nm
Nalive transferrin
I5 groups modified
17 groups modred
o.050 o.o41 0.034 0.034 0.027 O.Ol 7 0.009
o.047 0.025 0.027 0.027 0.026 o.o21 o.ol 5
0.047 0.026 0.023 0.025 0.023 -O.Ol 5
Iron-binding properties of modified transferrin preparations The iron-binding activity of transferrin as a function of various degrees of modification is shown in Fig. 2. It may be noticed that no change in iron-binding activity occurred until some 15 carboxyl groups had been modified. Then the ironbinding activity fell gradually until no binding occurred at an extrapolated value of 62 earboxyl groups modified. The stability of the iron-transferrin complex in the native and modified transferrins is shown in Table II. Transferrin samples with 15-17 earboxyl groups modified were used in these experiments. Such transferrin samples, judging from their A46~ nm/ A280 nm ratios, were still fully active (see Fig. 2). It may be seen from Table II that, whereas native transferrin lost its pink color gradually with increasing EDTA concentrations, the modified transferrin was 50% decolorized at the lowest EDTA concentration used (0.0025 M). The apparent half-saturation level was maintained in the presence of EDTA concentrations of o.oo25-O.Ol M, with a further loss of color taking place at EDTA concentrations of 0.025 and 0.05 M.
TABLE
II[
SEDIMENTATION
AND
OPTICAL
ROTATION
OF
NATIVI*2 A N D
MODIFIED
TRANSFERRIN
PREPARATIONS
All a n a l y s e s w e r e p e r f o r m e d w i t h i r o n - s a t u r a t e d s p e c i e s , r a n g i n g f r o m f u l l y a c t i v e t o i n a c t i v e p r e p a r a t i o n s . S o l v e n t A w a s 2.8 • i o -~ M F e a+ s t a b i l i z e d b y c i t r a t e in o . i M N a H C O a, w i t h a f i n a l p H 9.o. S o l v e n t B w a s o . i M s o d i u m a c e t a t e - a c e t i c a c i d a t p H 5.o.
N u m b e r of carboxyl groups modified
Solvent
Protein concn, (mg/ml)
Sedimentation coeffcient ( S units)
Specific rotation at 589 n m
o o o 15 17 3° 44 5°
A A B A A A A B
io.o 5.0 5.0 IO.O 5.0 5 .0 3-3 5.o
4.1 .5.0 4.8 4.1 4.,q 4-9 5.0 4.2
55 ° 55 ° --55 ° --49 ° --55 ° --52° --58° 53 °
Biochim. Biophys. Acta, 2I 4 ( t 9 7 o) 3 7 - 4 3
42
A. BEZKOROVAINY, D. GROHLICH
Physical properties of modified transferrins The modified transferrins retained their solubility at pH 7-9 if fewer than 35 carboxyl groups were modified. The solubility of this protein then decreased gradually with increasing degrees of modification, until it became completely insoluble with some 50 carboxyl groups substituted by glycine methyl ester. Such preparations were, however, perfectly soluble at pH 4 5The ultracentrifugal and optical rotatory properties of modified transferrins are given in Table I I I . It m a y be observed that no detectable changes in these parameters took place upon the modification of 35 4 ° carboxyl groups per molecule of transferrin. With 50 carboxyl groups modified, however, transferrin showed a significantly lower sedimentation coefficient compared to the native protein. The absorption spectrum of iron-saturated native and modified (16 carboxyl groups substituted) transferrins is shown in Fig. 3. It m a y be observed that the maxi-
0,30
0.28
i
0.26
o
0.21
0.22
420
440
480
480
500
;~Cnm) Fig. 3. Absorption spectra of native (Curve B) and modified (Curve A, 1G carboxyl groups modified) transferrins s a t u r a t e d with iron. Concentrations in b o t h cases were 5 mg/ml.
m u m of the modified protein is slightly shifted toward the blue region and is much broader than that of the native protein. The absorption at 465 nm was, however, almost identical for both proteins. DISCUSSION
Electrophoretic studies indicated that the mobility toward the positive pole of transferrin treated with glycine methyl ester and CMC is markedly retarded. It is unlikely that this phenomenon is due to any reason other than the blockage of carboxyl groups. The introduction of a large number of glycine residues into transferrin clearly results in the loss of its iron-binding activity. Since the loss of this activity took place over a wide range (I6-5o + carboxyl groups) rather than within a limited number Biochim. Bioph~/s. Acta, 2I 4 (I97 o) 37-43
M O D I F I C A T I O N OF T R A N S F E R R 1 N
4~
of carboxyl groups modified, it appears that the loss of activity was not due to the modification of specific iron-binding ligands. Instead, it appears that the gradual loss of activity was due to some sort of a limited conformational change in the transferrin molecule, brought about, perhaps, by the progressive disappearance of negative charges from the surface of the protein. Judging from the ultracentrifugal and polarimetric data, this conformational change apparently did not involve a gross alteration in the tertiary structure of the protein. A detectable conformational change was, however, seen with samples that had lost their ability to dissolve in neutral aqueous buffer solutions. The fact that the modification of some 5o carboxyl groups did result in the complete insolubilization and a conformational change in the transferrin molecule indicates that the carboxyl groups play an important role in the stabilization of transferrin under physiological conditions. Competition of transferrin and EDTA for iron showed that the ability of transferrin to bind iron is inversely proportional to the logarithm of EDTA concentration. This fact indicates that the loss of iron from transferrin in the presence of EDTA is random. A behavior different from that seen with the native transferrin was observed with the modified protein. Low cencentrations of EDTA were able to bring about a 50°o loss of color. We interpret these results as follows: the modification of some 16 carboxyl groups of transferrin results in the destabilization of one of the two ironbinding sites. This occurs because the more reactive carboxyl groups of transferrin are situated in its vicinity. The other binding site remains unaffected, perhaps because there are fewer reactive carboxyl groups in its vicinity. The shift of the spectrum maximum of the modified transferrin toward the blue range may indicate the exposure of the affected iron-binding site to the environment, i.e. its destabilization, thus supporting the results of the EDTA experiment. This work presents further evidence that the environments of the two ironbinding sites of transferrin are not identical. Such a view was expressed in a previous publication from this laboratory 2 which was based on the reactivities of tyrosyl residues of transferrin toward tetranitromethane. ACKNOWLEDGMENT
This work was supported by Grant No. 11985-07 of the U.S. National Institutes of Health. RI~FERENCES
I 2 3 4 5 6 7 8 9
J. J. \¥INDLE, A. K. WIERSMA, J. [{. CLARK AND R. E. FEENEY, Biochemistry, 2 (T963) 1341. \V. F. LINE, D . GROHLICH AND A. ~EZKOROVAINY, Biochemistry, 6 ( I 9 6 7 ) 3 3 9 3 . A. T. TAN AND R. C. V~TOOI)WORTH, Biochemistry, 8 (1969) 3711. S. I4. KOMATSU AND ]~. •. FEENEY, Biochemistry, 6 (1967) 1 1 3 6 . D. G. HOARE AND D. E. KOSHLAND, J R . , .]. Biol. Chem., 242 (1967) 2 4 4 7 . S. I~ORNFELD, Biochemistry, 7 (1968) 945. T. H . BOTHWELL, P. JACOBS AND J. D. TORRENCE, S. A f t . ,]. 1l'Ied. Sci., 27 (I962} 35. S. MOORE, D. H . SPACKMA.N AND W . H . STEIN, Anal. Chem., 3 ° (I958) I I 8 5 . H . E. SCHULTZE AND J. F. t{EREMANS, Molecular Biology of Human Proteins, E l s e v i e r , A m s t e r d a m , 1 9 6 6 , Vol. I, p. 212.
Biochim. Biophys. ,4cla, 2I 4 (197 o) 37--43
37
BBA 35612 MODIFICATION OF CARBOXYL GROUPS OF T R A N S F E R R I N
ANATOLY BEZKOROVAINY AND DIETMAR GI{OHLICH Biochemistry Department, Rz~sh-Presbyterian-St. Luke's 3Iedical Center, Chicago, Ill., 60612, and Department of Biological Chemist~iv, University of Illinois College of Medicine, Chicago, Ill., 6o6z2 (U.S.A.)
(Received March I6th, I97o)
SUMMARY Carboxyl groups of transferrin were coupled with glycine methyl ester using a water-soluble carbodiimide. The iron-binding activity of the modified protein did not change until some 16 carboxyl groups had been modified. The activity then fell in proportion to the number of groups modified until no iron binding was seen with 62 carboxyl groups modified. Such a slow drop in activity was interpreted to indicate that the carboxyl groups do not directly participate in the iron-binding reaction and that the activity was lost because of a conformational change in the protein structure. Experiments involving competition of native transferrin and EDTA foi iron indicated that the iron-binding activity of the former declined with increasing EDTA concentrations in a gradual manner. Similar experiments with transferrin with 15-17 carboxyl groups blocked by the glycine methyl ester showed that half of the iron-binding activity was lost with very low EDTA concentrations, remained at the 50% level over an EDTA concentration range of 0.0025 o.oi M and then commenced to decline in a gradual manner. It was concluded that the more reactive carboxyl groups are situated in the vicinity of only one of the two iron-binding sites of transferrin and that their modification then destabilizes the coordination complex. Very few if any reactive carboxyl groups are situated in the vicinity of the other iron-binding site. This finding supports the idea that the environments of the two iron-binding sites of transferrin are not identical.
INTRODUCTION Transferrin, the iron-binding protein of human serum, is said to bind two atoms of iron for each protein molecule. Each iron-binding site has been assumed to consist of two histidyl residues, 2-3 tyrosyl residues, and one bicarbonate ion 1. Whereas the binding of iron by nitrogen atoms has been established by both electron spin resonance 1 and chemical modification 2 studies, the role of tyrosyl residues has been surmised largely through indirect means, such as spectrophotometric titration data a and Abbreviation: CMC, I-cyclohexyl-3(2-morpholino-4-ethyl) carbodiimide methyl p-toluene sulfonate. Biochim. Biophys. Acla, zl 4 (t97o) 37 43
38
A. BEZKOROVAINY, D. GROHLICH
chemical modification w o r k 2,4. I t is t h u s i m p o r t a n t to i n v e s t i g a t e the possibility of the p a r t i c i p a t i o n of other a m i n o acid side chains in the iron-binding reaction. One such v e r y likely possibility are t h e c a r b o x y l groups, which, as c o m p o n e n t s of o t h e r compounds, are k n o w n to complex w i t h iron to form coordination derivatives. The purpose of the present c o m m u n i c a t i o n is to i n v e s t i g a t e the role of c a r b o x y l groups in the b i n d i n g of iron b y transferrin and their p a r t i c i p a t i o n in the m a i n t e n a n c e of the t e r t i a r y s t r u c t u r e of this protein. MATERIALS AND METHODS Chemicals The transferrin used in this w o r k was p u r c h a s e d from Hoechst L a b o r a t o r i e s ( W o o d b u r y , N.Y.). I t s molecular weight was assumed to be 83 ooo. Glycine m e t h y l ester hydrochloride a n d I-cyclohexyl-3(2-morpholino-4-ethyl) c a r b o d i i m i d e m e t h y l p-toluene sulfonate (CMC) were p u r c h a s e d from K a n d K L a b o r a t o r i e s (Plainview, N.Y.). All other chemicals were of reagent grade p u r i t y a n d were o b t a i n e d from F i s h e r Scientific Co. (Chicago, Ill.). Methods The coupling of t l a n s f e r r i n with glycine m e t h y l ester was done at p H 4.75 at 25 ° in an a u t o m a t i c t i t r a t i o n device according to the m e t h o d of HOARE AND KOSHLAND5. The c o n c e n t r a t i o n of CMC was v a r i e d for the purpose of o b t a i n i n g various degrees of modification of transferrin. A t y p i c a l e x p e r i m e n t was done as follows: 1.68 g of glycine m e t h y l ester-HC1 was dissolved in 7.5 ml water. The p H was a d j u s t e d to 4.75 with 5 M N a O H , a n d lOO-15o m g transferrin were then dissolved in this solution. A t zero t i m e O.l-O.4 g solid CMC was added, the reaction m i x t u r e was m a i n t a i n e d at p H 4.75, and 2-ml aliquots were w i t h d r a w n at various t i m e intervals (2-24o rain) a n d p i p e t t e d into 5 ml of o.5 M a c e t a t e buffer at p H 5.o to s t o p the reaction. These were t h e n d i a l y z e d against w a t e r a n d lyophilized. Controls were done b y i n c u b a t i n g all c o m p o n e n t s as described above, b u t o m i t t i n g the CMC. I r o n - b i n d i n g analyses of transferrins were p e r f o r m e d as follows: 5 mg of the n a t i v e or modified transferrin was weighed into a 13 m m × IOO m m test tube. To the p r o t e i n was then a d d e d I ml of a solution containing excess iron (this solvent was p r e p a r e d b y weighing out a p o r t i o n of F e N H 4 ( S Q ) 2, a d d i n g a sufficient volume of O.I M N a H C O 3 to m a k e a f i n a l Fe 3+ concentration of 2.8 .lO 4 M, a n d then a d d i n g a q u a n t i t y of solid t r i - s o d i u m c i t r a t e to just bring the iron into solution), or to the 5 m g p r o t e i n was a d d e d 0.88 ml w a t e r followed b y o. 12 ml of I • lO -3 M Fe 3+ in o.I M N a H C O a {prepared as above). The l a t t e r procedure p r o v i d e d a stoichiometric a m o u n t of iron. The solutions were p e r m i t t e d to s t a n d for I h at room t e m p e r a t u r e a n d their absorbbances were then m e a s u r e d at 465 n m a n d 280 n m (I : IO dilution). The iron-binding c a p a c i t y of the transferrin samples was then expressed as a r a t i o of t h e a b s o r b a n c e at 465 nm and 280 n m (ref. 6). F u l l y active transferrin h a d an A~65nm/A2sonm r a t i o of o.o48 o.o5o, whereas fully i n a c t i v e transferrin was assumed to have the A46 a nm/ ./128o nm r a t i o at o. In some instances it was necessary to c o m p a r e the s t a b i l i t y of the iron-transferrin complex in the modified p r o t e i n a n d n a t i v e protein. This was accomplished b y perm i t t i n g the transferrin p r e p a r a t i o n s to c o m p e t e for iron with various q u a n t i t i e s of Biochim. Biophys. Acta, 214 (197° ) 37-43
MODIFICATION
OF TRANSFERRIN
3 ()
ethylenediamine tetraacetic acid (EDTA) at pH 7.4. The procedure used was a modification of that described by BOTHWELL et al.V: 5-mg portions of transferrin were weighed into 13 m m × IOO mm test tubes. To each tube was added I ml of o.oo25-o.o5 M EDTA adjusted to pH 7.4. This was then followed by o.12 ml of the I .IO -3 M l"e~÷ solution (see above). The reaction mixtures were then placed in a water bath at 37 ° for I h, after which the A46~ nm/A280 nm ratios were determined.
Analytical procedures The amount of glycine incorporated into transferrin was measured via the automatic amino acid analyzer. The Spinco Model I2oC instrument was used according to the procedure of MOORE et al. 8. Prior to the analysis, the transferrin sample was hydrolyzed with 5.7 M HC1 for 24 h at IiO ° in a sealed tube previously flushed with nitrogen. The sample was repeatedly lyophilized after the hydrolysis. The number of carboxyl groups modified was determined by subtracting the number of intrinsic glycine residues in transferrin (50 as given by SCHULTZEngI) HEREMANS9 for a particle with a molecular weight of 83 ooo) from the total number of glycine residues present in a given sample. The precision of the amino acid analyses was approx. 5 %All speetrophotometric measurements were done in a Zeiss PMQ II instrument using the I-ml semi-micro cells. Optical rotation measurements were done in a Perkin Elmer Model 141 apparatus at 25 ° and 589 nm using the i-ml cells (I dm). Ultracentrifugal analyses were done in a Spinco Model E apparatus at 2o ° at full speed. Moving-boundary eleetrophoresis was performed in an Antweiler mieroelectrophoresis apparatus at 60 V (1. 4 mA) in the 0.05 M veronal buffer at pH 8.6. Proteins were analyzed at concentrations of 20 mg/ml. RESULTS
1"he incorporation of glycine into tran@rrin Different amounts ofglycine could be incorporated into transferrin by (a) varying the concentration of CMC in the reaction mixtures and (b) varying the time of reaction. The results of such experiments are shown in Table I. It may be noted that the')lighest number of glycine residues incorporated into transferrin was 54 per protein molecule. TA BLE
I
INCORPORATION
Data
OF GLYCINE
are numbers
Time of reaction
INTO
TRANSFERRIN
of glycine residues
incorporated
per molecule
of transferrin
C M C conch. (g/±o ml reaction m i x t u r e ) ;
(rain) o.I 2 5 15 3° 6o 90 240
o.2
7 16
29 17
0.3 i~ 21 37 39 44 --
0.4
27 ----
54
13iochim. Biophys. qcta, 2 t 4 (~967) 3 7 - 4 3
40
A. BEZKOROVAINY, D. GROHLICH
Fig. i. Moving-boundary electrophoresis of native and modified transferrins. I n b o t h cases the p h o t o g r a p h s were taken 4 ° rain after commencing the run. A. Native transferrin. B. Transferrin with 3 ° carboxyl groups modified.
Modified and native transferrin samples were analyzed by the method of movingboundary electrophoresis. In general, the mobilities of modified transferrins were retarded in proportion to the number of glycine groups introduced. A representative electrophoresis pattern is shown in Fig. i.
0.05
O~ O -,
O.OSL~ A46,,~S 0.04 I~O~o A280 0,03
O~
0.04
A465
o
O
0,03 i
A280 0.02
~
O
~
o
IL0 20 30 40 50 60 TiME (min) ~ O
0.01 I
I
I
J
10
20
30
40
NUMBER OF GLYCINE R E S I D U E S I N T R O D U C E D T R A N S F E R R I N MOLECULE
50
60
I N T O EACH
Fig. 2. Iron-binding activity of transferrin as a function of the n u m b e r of carboxyl groups modified. The inset g r a p h presents the rate of transferrin inactivation in the presence of o.o 7 31 CMC
Biochim. Biophys. Acta, 214 (197 o) 37 43
MODIFICATION OF TRANSFERRIN TABLE
41
II
COMPETITION
OF
tDWa
Protein concentrations
E D T A concn. (M)
o 0.0025 0.0050 0.0075 O.OLOO 0.0250 0.0500
WITH
VARIOUS
TRANSFERRIN
PREPARATIONS
FOR
IRON
w e r e 5 m g / m l in all c a s e s .
A46~nrn/A~ s o
nm
Nalive transferrin
I5 groups modified
17 groups modred
o.050 o.o41 0.034 0.034 0.027 O.Ol 7 0.009
o.047 0.025 0.027 0.027 0.026 o.o21 o.ol 5
0.047 0.026 0.023 0.025 0.023 -O.Ol 5
Iron-binding properties of modified transferrin preparations The iron-binding activity of transferrin as a function of various degrees of modification is shown in Fig. 2. It may be noticed that no change in iron-binding activity occurred until some 15 carboxyl groups had been modified. Then the ironbinding activity fell gradually until no binding occurred at an extrapolated value of 62 earboxyl groups modified. The stability of the iron-transferrin complex in the native and modified transferrins is shown in Table II. Transferrin samples with 15-17 earboxyl groups modified were used in these experiments. Such transferrin samples, judging from their A46~ nm/ A280 nm ratios, were still fully active (see Fig. 2). It may be seen from Table II that, whereas native transferrin lost its pink color gradually with increasing EDTA concentrations, the modified transferrin was 50% decolorized at the lowest EDTA concentration used (0.0025 M). The apparent half-saturation level was maintained in the presence of EDTA concentrations of o.oo25-O.Ol M, with a further loss of color taking place at EDTA concentrations of 0.025 and 0.05 M.
TABLE
II[
SEDIMENTATION
AND
OPTICAL
ROTATION
OF
NATIVI*2 A N D
MODIFIED
TRANSFERRIN
PREPARATIONS
All a n a l y s e s w e r e p e r f o r m e d w i t h i r o n - s a t u r a t e d s p e c i e s , r a n g i n g f r o m f u l l y a c t i v e t o i n a c t i v e p r e p a r a t i o n s . S o l v e n t A w a s 2.8 • i o -~ M F e a+ s t a b i l i z e d b y c i t r a t e in o . i M N a H C O a, w i t h a f i n a l p H 9.o. S o l v e n t B w a s o . i M s o d i u m a c e t a t e - a c e t i c a c i d a t p H 5.o.
N u m b e r of carboxyl groups modified
Solvent
Protein concn, (mg/ml)
Sedimentation coeffcient ( S units)
Specific rotation at 589 n m
o o o 15 17 3° 44 5°
A A B A A A A B
io.o 5.0 5.0 IO.O 5.0 5 .0 3-3 5.o
4.1 .5.0 4.8 4.1 4.,q 4-9 5.0 4.2
55 ° 55 ° --55 ° --49 ° --55 ° --52° --58° 53 °
Biochim. Biophys. Acta, 2I 4 ( t 9 7 o) 3 7 - 4 3
42
A. BEZKOROVAINY, D. GROHLICH
Physical properties of modified transferrins The modified transferrins retained their solubility at pH 7-9 if fewer than 35 carboxyl groups were modified. The solubility of this protein then decreased gradually with increasing degrees of modification, until it became completely insoluble with some 50 carboxyl groups substituted by glycine methyl ester. Such preparations were, however, perfectly soluble at pH 4 5The ultracentrifugal and optical rotatory properties of modified transferrins are given in Table I I I . It m a y be observed that no detectable changes in these parameters took place upon the modification of 35 4 ° carboxyl groups per molecule of transferrin. With 50 carboxyl groups modified, however, transferrin showed a significantly lower sedimentation coefficient compared to the native protein. The absorption spectrum of iron-saturated native and modified (16 carboxyl groups substituted) transferrins is shown in Fig. 3. It m a y be observed that the maxi-
0,30
0.28
i
0.26
o
0.21
0.22
420
440
480
480
500
;~Cnm) Fig. 3. Absorption spectra of native (Curve B) and modified (Curve A, 1G carboxyl groups modified) transferrins s a t u r a t e d with iron. Concentrations in b o t h cases were 5 mg/ml.
m u m of the modified protein is slightly shifted toward the blue region and is much broader than that of the native protein. The absorption at 465 nm was, however, almost identical for both proteins. DISCUSSION
Electrophoretic studies indicated that the mobility toward the positive pole of transferrin treated with glycine methyl ester and CMC is markedly retarded. It is unlikely that this phenomenon is due to any reason other than the blockage of carboxyl groups. The introduction of a large number of glycine residues into transferrin clearly results in the loss of its iron-binding activity. Since the loss of this activity took place over a wide range (I6-5o + carboxyl groups) rather than within a limited number Biochim. Bioph~/s. Acta, 2I 4 (I97 o) 37-43
M O D I F I C A T I O N OF T R A N S F E R R 1 N
4~
of carboxyl groups modified, it appears that the loss of activity was not due to the modification of specific iron-binding ligands. Instead, it appears that the gradual loss of activity was due to some sort of a limited conformational change in the transferrin molecule, brought about, perhaps, by the progressive disappearance of negative charges from the surface of the protein. Judging from the ultracentrifugal and polarimetric data, this conformational change apparently did not involve a gross alteration in the tertiary structure of the protein. A detectable conformational change was, however, seen with samples that had lost their ability to dissolve in neutral aqueous buffer solutions. The fact that the modification of some 5o carboxyl groups did result in the complete insolubilization and a conformational change in the transferrin molecule indicates that the carboxyl groups play an important role in the stabilization of transferrin under physiological conditions. Competition of transferrin and EDTA for iron showed that the ability of transferrin to bind iron is inversely proportional to the logarithm of EDTA concentration. This fact indicates that the loss of iron from transferrin in the presence of EDTA is random. A behavior different from that seen with the native transferrin was observed with the modified protein. Low cencentrations of EDTA were able to bring about a 50°o loss of color. We interpret these results as follows: the modification of some 16 carboxyl groups of transferrin results in the destabilization of one of the two ironbinding sites. This occurs because the more reactive carboxyl groups of transferrin are situated in its vicinity. The other binding site remains unaffected, perhaps because there are fewer reactive carboxyl groups in its vicinity. The shift of the spectrum maximum of the modified transferrin toward the blue range may indicate the exposure of the affected iron-binding site to the environment, i.e. its destabilization, thus supporting the results of the EDTA experiment. This work presents further evidence that the environments of the two ironbinding sites of transferrin are not identical. Such a view was expressed in a previous publication from this laboratory 2 which was based on the reactivities of tyrosyl residues of transferrin toward tetranitromethane. ACKNOWLEDGMENT
This work was supported by Grant No. 11985-07 of the U.S. National Institutes of Health. RI~FERENCES
I 2 3 4 5 6 7 8 9
J. J. \¥INDLE, A. K. WIERSMA, J. [{. CLARK AND R. E. FEENEY, Biochemistry, 2 (T963) 1341. \V. F. LINE, D . GROHLICH AND A. ~EZKOROVAINY, Biochemistry, 6 ( I 9 6 7 ) 3 3 9 3 . A. T. TAN AND R. C. V~TOOI)WORTH, Biochemistry, 8 (1969) 3711. S. I4. KOMATSU AND ]~. •. FEENEY, Biochemistry, 6 (1967) 1 1 3 6 . D. G. HOARE AND D. E. KOSHLAND, J R . , .]. Biol. Chem., 242 (1967) 2 4 4 7 . S. I~ORNFELD, Biochemistry, 7 (1968) 945. T. H . BOTHWELL, P. JACOBS AND J. D. TORRENCE, S. A f t . ,]. 1l'Ied. Sci., 27 (I962} 35. S. MOORE, D. H . SPACKMA.N AND W . H . STEIN, Anal. Chem., 3 ° (I958) I I 8 5 . H . E. SCHULTZE AND J. F. t{EREMANS, Molecular Biology of Human Proteins, E l s e v i e r , A m s t e r d a m , 1 9 6 6 , Vol. I, p. 212.
Biochim. Biophys. ,4cla, 2I 4 (197 o) 37--43