British Journal of Haematology, 1975,29,
511.
Differences between Human Fe,-Transferrin Molecules R. S . LANE Haernatology Departnzent, St George's Hospital, London (Received 17 June 1974; accepted f o r publication 8 August 1974) SUMMARY. Two molecular forms of Fe, -transferrin molecule can be demonstrated by anion-exchange chromatography on columns of DEAE-cellulose, pH 7.90. It is believed that co-ordination of each metal-binding site by ferric ions induces dissimilar changes in molecular conformation in the region of the binding sites which, in the case of one site, rcsults in the molecule behaving as a weaker anion during anion-exchange chromatography. If so, this could be entirely in accord with current views that the two binding sites of transferrin do not share equal properties of iron exchange with cells. At physiological levels of transferrin iron-saturation, it is likely that two populations of Fe,-transferrin molecules form the bulk of the iron-transfcrriii complex. Transferrin is a controlling factor in iron metabolism and is responsible for the specific transfer of ferric ions to developing erythroid cells; however, the molecular basis for the physiological role of transferrin has yet to be determined. As Aisen recounts (1974), transferrin has two metal-binding sites which have similar physical properties : small differences bctween the two binding sites have been described but these have been demonstrated mostly in non-physiological experimental conditions. Even so, such findings heighten the interest in the data produced by Fletcher & Huehns (1967, 1968) suggcsting that the two binding sites of transferrin have unequal potential to release iron to cells. Human iron-transferrin complex can be separated from apo-transferrin on columns of DEAE-cellulose at pH 7.90 by using an ascending concentration gradient of tris-hydrochloric acid (Lane, 1971). It is now evident that, with this system, two forms of Fe,-transferrin (transferrin with iron bound at only one metal-binding site) are separated depending upon which binding site is co-ordinated by the ferric ion. These findings are now presented. MATERIALS AND METHODS Isolation and purification of human transferrin C from pooled serum and the chromatography procedure on columns of DEAE-cellulose have been described elsewhere (Lane, 1971). For the elution of transferrin from DEAE-cellulose, 0.9 x 60 cm columns of Whatman precycled DE-52 cellulose were equilibrated with starting buffer at a constant flow rate of 20 ml/h. Starting buffer was metal-free tris-HC1, pH 7.90, conductance 2.0 mmho at 25'C. From these columns, transferrin was eluted with an increasing concentration gradient of buffer salt, obtained by drawing into a fixed 250 ml volume of starting buffer a solution Correspondence: Dr R. S. Lane, North East Metropolitan Blood Transfusion Service, Brentwood, Essex.
SII
R.S . Lane
512
of tris-HC1 having a concentration four times that of the starting buffer. Equilibration and elution were controlled by measuring pH and conductance at 25OC. The following procedure was used for binding iron to transferrin in order that conditions were optimal for the random uptake of iron at either metal-binding site of the protein. Human apo-transferrin in tris-HC1 starting buffer was gently stirred in a beaker. A trace of bicarbonate was added and the mixture titrated to pH 5.0 with M HC1: to this solution was added sufficient59Fe in 10 mM HC1 to achieve the required iron saturation of transferrin. Then the mixture was back-titrated to pH 7.90 with M NaOH. Iron was rapidly bound by transferrin between pH 6.5 and 7.0 and the complex was then dialysed at 4’C against trisHCl starting buffer. After dialysis, protein concentration and 59Fe-binding to transferrin were checked by determining optical density of the solution of 280 nm and 465 nm respectively and by measuring 59Feactivity in the sample. Transferrin samples at various levels of iron-saturation were chromatographed on columns of DEAE-cellulose. In each eluted fraction 59Fe activity was measured with appropriate standards in a Packard Autogamma Spectrometer and from the specific activity of 59Fethe iron content was calculated. To estimate transferrin concentration, the value for E i L at 280 nm was taken as 10.4 for human apo-transferrin. (For this purpose, the iron-transferrin complex was dissociated with a small aliquot of HCl added to each fraction: the effect of this volume on transferrin concentration was very small and was ignored.) Total iron binding capacity (TIBC) of each fraction was calculated from transferrin concentration, assuming
Fraction
FIG I
Fraction
FIG z
FIG I . DEAE-cellulose chromatography of human iron-free transferrin. Iron-free transferrin (Tf) was eluted as a single symmetrical peak. FIG 2. DEAE-cellulose chromatography of human transferrin (Tf), sample B: transferrin was 15% satured with 59Fe. Both Tf and Tf-bound 59Feactivity were eluted as bifid peaks; the smaller Tf peak eluted at fraction 64 was seen to coincide with the first peak of Tf-bound 59Fe.
TransfewinMolecular Diyerences
513
the molecular weight of transferrin to cqual 80 000. From the total iron content and TIBC, iron saturation of transferrin in each fraction was determined.
RESULTS The level of iron saturation of transferrin samples used in the experiments described below and the aniount of transferrin used on each colunxi is shown in Table I. Fig I shows the elution of iron-free transferrin from sample A which was eluted as a symmetrical peak betwcen fractions 60 and 80: the volume of eluted fractions was 3.5 ml. In four experiments, mean buffer conductance at peak protein concentration was 4.94+ 0.02 mmho at 250C. TABLE I. The level of iron saturation of transferrin in samples A-F and the amount of transferrin applied to each DEAE-cellulose column
I
Sample
A ~
B ~
:/, iron saturation mg transferrin/column
_
_
~
_
0
IS
21
40.4
IS
15
30
30
1
Sarnole C
E
D
C _
F
_ _ 58.5 30
_
_
_
I00
30
Sornple D
\
Tf -bound
/”Fe I
40
50
40
60
50
60
Froc tion
FIG 3. DEAE-cellulose chromatography of human transferrin (Tf).Iron saturation in samples C and D was 21% and 40.4% respectively. With increasing iron saturation of transferrin, the elution profile of Tf became more obviously bifid, the first eluted Tf peak increasing in size and being associated with the main peak of Tf-bound 59Feactivity.
_
R. S. Lane
514
Fig 2 (sample B) shows the optical density at 280 nm of the eluted transferrin (Tf) and the iron-transferrin complex (Tf-bound 59Fe)obtained from the 59Feactivity in each fraction. Fraction volume was 3.5 ml. Tf was eluted between fractions 60 and 80 and the profile was bifid; a small peak was seen at fraction 64 and the main peak at fraction 69. Tf-bound 59Fe also showed two peaks, the first at fraction 65 was slightly larger and coincided with the first peak of Tf; the second Tf-bound 59Fepeak was at fraction 74, that is, after the main Tf peak. Fig 3 shows the elution profiles of Tf and Tf-bound 59Fefrom samples C and D. In these experiments, the volume of each eluted fraction was 5.6 ml which accounts for the lower fraction numbers at which transferrin was eluted from D E A E - c ~ ~ ~columns. u~os~ Transferrin in sample C was 21% saturated with iron. Tf was eluted between fractions 40 and 60; peak concentration was at fraction 45 and a distinct shoulder to the main Tf peak was seen at fractions 42/43. As in sample B, Tf-bound 59Fefrom sample C showed two peaks, the first peak at fraction 43 was larger and coincided with the shoulder to the Tf peak; the second peak at fraction 49 was eluted after the main Tf peak. At the higher iron saturation of transferrin in sample C, the first eluted peak of Tf-bound 59Fe showed the relatively greater increase in size. Comparing the results of samples B and C, the greater definition of the Tf profile from sample B was obtained by the use of only 15 mg transferrin on the column and by collecting
Somple E
Fraction
FIG 4. DEAE-cellulose chromatography of human transferrin (Tf). Iron saturation in samples E and F was 58.5% and 100% respectively. In sample E, the first peak of Tf with its associated peak of Tfbound 59Feshowed that this sample contained more iron-complexed than iron-free transferrin. Sample F represented the elution of homogeneous Fe2-transferrin molecules which were eluted as a single homogeneous peak. In this sample the second peak of Tf-bound 59Fe was absent.
Transferrin Molecular DiJerences
51s
a smaller fraction volume. The use of 3 0 mg of transferrin and the collection of larger fractions in samples C-F, however, did allow more convenient manipulation of eluted samples. Transferrin in sample D was 40.4% saturated with iron. Tf was eluted in two peaks at fractions 43 and 47/48. Tf-bound 59Fe also showed two peaks: the first, at fraction 44, coincided with the first Tf peak, while the second, at fraction 51, characteristically followed the second peak of Tf. Compared with sample C, sample D contained twice as much iron. The bifid nature of the Tf peak was obvious and the association between the first peaks of Tf and Tf-bound 59Fereadily apparent. The increased amount of iron-transferrin complex in sample D presented mainly in the first Tf-bound 59Fepeak. Fig 4 shows the elution profiles of Tf and Tf-bound 59Fefrom samples E and F. Transferrin in sample E was 58.5% saturated with iron; there were two TEpeaks of which the first was larger and its association with the first peak of Tf-bound 59Feshowed that sample E contained more iron-complexed than iron-free transfcrrin. Equally important, the second peaks of Tf-bound 59Fein both samples D and E were approximately equal in size, thus this peak was maximal when initial iron-saturation in the transferrin sample approximated to 50%. In sample F, single peaks of Tf and Tf-bound 59Fewere seen together at fraction 43 and represented the elution of homogeneous Fez-transferrin molecules. In four experiments with iron-saturated transferrin, carried out in the manner used for apo-transferrin in sample A, peak elution of Fez-transferrin occurred at a mean level of buffer conductance of 4 . p + 0.01mmho at z5OC. DISCUSSION If iron binds with equal readiness at either binding site of transferrin (Aasa et al, 1963),then the proportions of apo-transferrin, Fe,-transferrin and Fez-transferrin in a transferrin solution of known saturation (S%) can be determined since the probability of an iron atom binding at any one site is S/IOO. It follows then: (100- S)2
Apo-transferrin Fe, -transferrin Fez-transferrin
I00
%
2s (100- S)
I00
%
S2 -% I00
Accordingly, the distribution of transferrin molecules in samples B-E are shown in Table 11. As the iron saturation in a transferrin sample increased from o to IOO%, the single peak of apo-transferrin was progressively replaced by two and then finally one peak of iron-transferrin complex. In the I 5 % iron-saturated sample B, Fez-transferrin molecules were negligible in number, yet two approximately equal peaks of iron-transferrin complex were eluted which strongly suggested that they contained equal populations of Fe,-transferrin molecules. As iron content in the transferrin samples increased so did their content of Fe,-transferrin molecules. Chromatographically, this became apparent as an increase in the first eluted peak
R. S. Lane
516
TABLE 11. The distribution of apo-transferrin, Fel-transferrin and Fez-transferrin molecules in samples B, C, D and E used for DEAE-cellulose chromatography Sample
Trangerrin iron-saturation
B C D E
% Apo-trangerrin
% Fel-transferrin
% Fe,-transferrin
molecules
molecules
molecules
72.25 62.4 35.5 17.2
25.5
15 21
40.4 58.5
2.25
33.2 48.2 48.6
4.4 16.3 34.2
TABLE 111. The level of iron-saturation and concentration of transferrin in selected fractions eluted during chromatography of samples C, D and E on DEAE-cellulose columns
Fraction
Sample C, 21%
iron-saturated
Sample D, 40.4%
iron-saturated
42 43 44 45 46 47 41 42 43 44 45 46 47 48 49 50
Sample E, 58.5%
iron-saturated
Transferrin (mglfracfion)
Iron saturation (%)
1.1
43 47 34
1.9 2.3 3.3 3.8 3.6 0.2
I2
6 3
2.3 2.6* 2.3
47 49 56 61 65
2.0
52
1.0
2.5
3.o 3.o 2.7
41 42 43 44 45 46 47 48 49
I .8 2.1
50
2.2
First Tf peak
'1 8
0.5
47
2.4 3.8* 3.7* 3.1 2.3 1.8
52
59 65 69 70 65 38231 16
Second Tf peak
First Tf peak Second Tf peak
* Fractions containing highest transferrin concentration within the first eluted peak of Tf in samples D and E.
Transfrrin Molecular Dtserences 517 of Tf-bound 59Fe which, in 100% iron-saturated transferrin, replaced both apo-transferrin and the second peak of Tf-bound 59Fe. Table I11 shows the levels of iron-saturation in transferrin fractions comprising the first and second peaks of Tf: within the first peak, fractions up to 43 were approximately 50% saturated and therefore these molecules were predominantly Fe, -transferrin. In samples D and E, fractions between 44 and 47 contained transferrin with levels of iron saturation approaching 70% and therefore these fractions contained a proportion of Fez-transferrin molecules. The distribution of iron saturation within the first Tf peak therefore showed that both Fe,-transferrin and Fez-transferrin molecules were present and that Fe,-transferrin was eluted first from DEAE-cellulose. In addition, Table I11 shows that the second Tf peak was mainly formed by apo-transferrin molecules. The iron status of transferrin molecules in the second peak of Tf-bound 59Fecould not be measured directly due to the proximity of the peak of apo-transferrin. However, other evidence indicated that this peak contained a second population of Fel-transferrin molecules : (I) Essentially, sample B was a mixture of apo-transferrin and Fel -transferrin molecules, yet two peaks of iron-transferrin complex were observed, of which the first was shown to contain Fe,-transferrin molecules. In sample B, both peaks of Tf-bound 59Fe were approximately the same in size, which would be expected in two populations of Fe,-transferrin molecules if iron atoms bind with equal facility at either metal-binding site of transferrin. (2) The maximum content of Fe, -transferrill molecules in transferrin samples occurs when net iron-saturation of the sample is 50%. Transferrin iron-saturation in sample D was 40.4% and in sample E was 58.5%, thus each sample contained equal proportions of Fe,-transferrin molecules. That the second peaks of Tf-bound 59Feeluted from samples D and E were equal in size, was consistent with these peaks containing Fe,-transferrin molecules. (3) In iron-saturated transferrin samples (Fez-transferrin), the second peak of Tf-bound 59Fewas absent: had this peak been attributable, not to Fel-transferrin, but to a more anodal transferrin variant or a proportion of partially denatured transferrin having a greater affinity for DEAE-cellulose than Fez-transferrin, then the second Tf-bound 9Fe peak would have persisted in the 100% iron-saturated transferrin sample. (4)It was assumed that iron in the first peak of iron-transferrin complex was attributable to half the sample population of Fe,-transferrin molecules and to the whole population of Fez-
TABLE IV. The amount of iron bound by transferrin in the first and second peaks of Tf-bound "Fe eluted from DEAE-cellulose columns. The first Tf-bound 59Fe peak contains both Fe,transferrin and Fe2-transferrin molecules. Iron bound by Fe,-transferrin within this peak is determined by subtraction and is shown to be equal to the iron bound by transferrin in the second Tf-bound 59Fepeak.
Sample
Iron saturation (%)
Total transferrinbound iron in -first peak (PA4
Iron infirst peak (pg)bound to Fe ,-trunsferrin
Iron in second peak ( p g ) of irontransferrin complex
B
IS
1.51
1.08
1.15
C
21
4.0
2.52
D
40.4
8.61
3.7
2.6 3.53
R.S . Lane
518
transferrin molecules; likewise iron in the second peak of iron-transferrin complex was attributed to a half-population of Fe,-transferrin molecules. Iron bound by Fe,-transferrin in each peak was determined and examples are given in Table IV. It is seen that equal amounts of iron were bound by Fe,-transferrin molecules in the two peaks of iron-transferrin complex, confirming the view that these peaks contained equal populations of Fe, -transferrin molecules. Fig 5 shows the relationship between chromatographic and the established electrophoretic behaviour of transferrin (Aisen et al, 1966) and the relative molecular charge of transferrin molecules. Electrophoretic behaviour correlates with net molecular charge whereas chromatographic behaviour does not : Fe, -transferrin and Fez-transferrin molecules, although more negatively charged, both behave as weaker anions than apo-transferrin on DEAEcellulose columns at pH 7.90; in addition, two forms of Fc,-transferrin molecule are identified by chromatography. For descriptive purposes, these molecules are termed Fe,-transferrin A and Fe,-transferrin B, where the former is eluted first from the column.
Increasing onionic affinity
- transferrin
DEAEcellulose: pH 7.90
Fe,
Electro phoresis : pH 8.40
Apo-transferrin
-
Fez transferrin
Apo - transferrin
Fe I - transferrin
Fe,
Fe, - transferrin
- tronsferrin
FIG 5. The relationship between the established electrophoretic behaviour of transferrin and its chromatographic behaviour on columns of DEAE-cellulose. The units of negative charge shown in Fet-transferrin and Fe2-transferrin molecules are relative to the molecular charge of apo-transferrin. Each unit of negative charge is due to the binding by transferrin of one bicarbonate ion with each atom of iron.
The physical properties of molecules which influence their electrophoretic mobility and affinity to anion-exchange media are different.The interaction between transferrin and DEAEcellulose is between reactive, charged, surface groups. This interaction i s complex, yet the equilibrium between buffer ions, protein, cellulose and DEAE groups is very susceptible to changes in charge, permitting sequential elution of molecules whose differences in reactive charge are small. Evidently, the surface reactive charge of Fe, -transferrin molecules is not uniform, half the molecular population exhibiting a significant decrease in electronegative characteristics. Such behaviour is best explained by relating it to the changes in molecular conformation that occurs in transferrin molecules when iron i s bound, Conformational change may be generalized, for example, affecting the hydrodynamic volume of transferrin (Charlwood, 1971) ; alternatively, there are changes localized in the region of the metal-binding sites, which Tan (1971) has suggested represent the major changes in conformation induced by iron binding. Charlwood has shown a stepwise re-
Transferrin Molecular Dixerences
519 duction in hydrodynamic volume of 0.7% in human transferrin after the binding of each iron atom and it seems improbable that this would permit chromatographic differentiation between two forms of Fe,-transferrin molecule. It is more likely that chromatographic behaviour of Fe,-transferrin molecules is caused by localized changes in conformation in the region of the binding sites. It is probable that the co-ordination of each metal-binding site by iron induces dissimilar changes in molecular conformation in the region of the sites: in the case of the binding site which combines with iron in Fe,-transferrin A, the resultant conformational change is associated with loss of a surface reactive charged group previously able to react with DEAE-cellulose. The exact explanation of these chromatographic results is not known due to lack of information about the molecular structure of transferrin. Decreased electronegative behaviour of Fel-transferrin A could result from an alteration in the reactivity of an a-carboxyl group which is negatively charged at pH 7.90. In support of this, under comparable experimental conditions, loss of a single charged sialic acid residue from transfcrrin causes a decrease in a&nity of transferrin for DEAE-cellulose similar to that caused by iron-binding in Fe,transferrin A (unpublished data). In addition, Bezkorovainy & Grohlich (1970) have provided evidence suggesting that reactivity of carboxyl groups is not the same in the region of both binding sites of human transferrin. The chromatographic data are consistent with other experimental evidence which shows differences between the metal-binding sites (Aisen et al, 1969; Aasa, 1972; Price & Gibson, 1972) and it is thought that the conditions of the chromatographic studies approach more nearly to the physiological environment than is often the case. The finding of two equal populations of Fel-transferrin molecules is in agreement with Aasa et a2 (1963) who described the binding sites of transferrin as independent and equivalent structures. It is of interest that in transferrin with iron saturation approaching the physiological range of 20-40%~ two populations of Fe,-transferrin molecules form the bulk of available irontransferrin complex. This could be entirely in accord with the views of Fletcher & Huehns (1968) that the properties of iron transport are not uniformly shared by all the metal-binding sites of transferrin. Could a difference in reactive charge associated with one metal-binding site have any influence 011 the process of iron transfer to cells? More than one factor is responsible for the release of iron from transferrin, but of interest is the fact that Bezkorovainy & Grohlich (1970) were able to chelate iron more readily from one binding site of transferrin after minimal chemical modification of reactive carboxyl groups on the molecule. In conclusion, co-ordination of the binding site in Fe,-transferrin A caused a significant reduction in affinity of transferrin for DEAE-celldose. Iron bound at the binding site in Fe,-transferrin B caused a minimal change in the chromatographic behaviour shown by apo-transferrin. Transferrin with iron bound at sites A and B (Fez-transferrin) behaved like Fe,-transferrin A on DEAE-cellulose columns, thus the charge effect associated with site A dominated the chromatographic behaviour of the molecule.
REFERENCES AASA, R. (1972) Reinterpretation of the electron paramagnetic resonance spectra of transferrins.
Biochemical and Biophysical Research Communications, 49s 806.
R.S. Lane AASA,R., MALMSTROM, B.G., SALTMAN, P.& VX”G ~ DT., (1963) The specific binding of iron(I1I) and copper(I1) to transferrin and conalbumin. Biochimica et Biophysica Acta, 75, 203. AISEN,P. (1974) The role of transferrin in iron transport. (Annotation). Britirh Journal of Haematology, 26, 159.
AISEN,P., AASA,R. & REDFIELD, A.G. (1969) The chromium, manganese, and cobalt complexes of transferrin. Journal qf Biological Chemistry, 2 4 , 4628.
AISEN,P., LEIBMAN, A. & REICH,H.A. (1966) Studies on the binding of iron to transferrin and conalbumin. Journal ofBiological Chemistry. 241, 1666. BEZKOROVAINY, A. & GROHLICH, D. (1970) Modification of carboxyl groups of transferrin. Biochimica et Biophysica Acta, 214,37. CHARLWOOD, P.A. (1971) Differential sedimentationvelocity and gel-filtration measurements on human
apotransferrin and iron-transferrin. Biochemical Journal, 125, 1019. FLETCHER, J. & HUEHNS,E.R. (1967) Significance of the binding of iron by transferrin. Nature, 215, 584. FLETCHER, J. & HUEHNS,E.R. (1968) Function of transferrin. Nature, 218, 1211. LANE,R.S. (1971) DEAE-cellulose chromatography of human transferrin: the effect of increasing iron saturation and copper(I1) binding. Biochimica et Biophysica Acta, 243, 193. PRICE,E.M. & GIBSON, J.F. (1972) Electron paramagnetic resonance evidence for a distinction between the two iron-binding sites in transferrin and in conalbumin. Journal of Biological Chemistry, 247, 8031.
TAN,A.-T. (1971) Circular dichroism properties of conalbumin and its iron and copper complexes. Canadian Journal of Biochemistry, 49, 1071.
511.
Differences between Human Fe,-Transferrin Molecules R. S . LANE Haernatology Departnzent, St George's Hospital, London (Received 17 June 1974; accepted f o r publication 8 August 1974) SUMMARY. Two molecular forms of Fe, -transferrin molecule can be demonstrated by anion-exchange chromatography on columns of DEAE-cellulose, pH 7.90. It is believed that co-ordination of each metal-binding site by ferric ions induces dissimilar changes in molecular conformation in the region of the binding sites which, in the case of one site, rcsults in the molecule behaving as a weaker anion during anion-exchange chromatography. If so, this could be entirely in accord with current views that the two binding sites of transferrin do not share equal properties of iron exchange with cells. At physiological levels of transferrin iron-saturation, it is likely that two populations of Fe,-transferrin molecules form the bulk of the iron-transfcrriii complex. Transferrin is a controlling factor in iron metabolism and is responsible for the specific transfer of ferric ions to developing erythroid cells; however, the molecular basis for the physiological role of transferrin has yet to be determined. As Aisen recounts (1974), transferrin has two metal-binding sites which have similar physical properties : small differences bctween the two binding sites have been described but these have been demonstrated mostly in non-physiological experimental conditions. Even so, such findings heighten the interest in the data produced by Fletcher & Huehns (1967, 1968) suggcsting that the two binding sites of transferrin have unequal potential to release iron to cells. Human iron-transferrin complex can be separated from apo-transferrin on columns of DEAE-cellulose at pH 7.90 by using an ascending concentration gradient of tris-hydrochloric acid (Lane, 1971). It is now evident that, with this system, two forms of Fe,-transferrin (transferrin with iron bound at only one metal-binding site) are separated depending upon which binding site is co-ordinated by the ferric ion. These findings are now presented. MATERIALS AND METHODS Isolation and purification of human transferrin C from pooled serum and the chromatography procedure on columns of DEAE-cellulose have been described elsewhere (Lane, 1971). For the elution of transferrin from DEAE-cellulose, 0.9 x 60 cm columns of Whatman precycled DE-52 cellulose were equilibrated with starting buffer at a constant flow rate of 20 ml/h. Starting buffer was metal-free tris-HC1, pH 7.90, conductance 2.0 mmho at 25'C. From these columns, transferrin was eluted with an increasing concentration gradient of buffer salt, obtained by drawing into a fixed 250 ml volume of starting buffer a solution Correspondence: Dr R. S. Lane, North East Metropolitan Blood Transfusion Service, Brentwood, Essex.
SII
R.S . Lane
512
of tris-HC1 having a concentration four times that of the starting buffer. Equilibration and elution were controlled by measuring pH and conductance at 25OC. The following procedure was used for binding iron to transferrin in order that conditions were optimal for the random uptake of iron at either metal-binding site of the protein. Human apo-transferrin in tris-HC1 starting buffer was gently stirred in a beaker. A trace of bicarbonate was added and the mixture titrated to pH 5.0 with M HC1: to this solution was added sufficient59Fe in 10 mM HC1 to achieve the required iron saturation of transferrin. Then the mixture was back-titrated to pH 7.90 with M NaOH. Iron was rapidly bound by transferrin between pH 6.5 and 7.0 and the complex was then dialysed at 4’C against trisHCl starting buffer. After dialysis, protein concentration and 59Fe-binding to transferrin were checked by determining optical density of the solution of 280 nm and 465 nm respectively and by measuring 59Feactivity in the sample. Transferrin samples at various levels of iron-saturation were chromatographed on columns of DEAE-cellulose. In each eluted fraction 59Fe activity was measured with appropriate standards in a Packard Autogamma Spectrometer and from the specific activity of 59Fethe iron content was calculated. To estimate transferrin concentration, the value for E i L at 280 nm was taken as 10.4 for human apo-transferrin. (For this purpose, the iron-transferrin complex was dissociated with a small aliquot of HCl added to each fraction: the effect of this volume on transferrin concentration was very small and was ignored.) Total iron binding capacity (TIBC) of each fraction was calculated from transferrin concentration, assuming
Fraction
FIG I
Fraction
FIG z
FIG I . DEAE-cellulose chromatography of human iron-free transferrin. Iron-free transferrin (Tf) was eluted as a single symmetrical peak. FIG 2. DEAE-cellulose chromatography of human transferrin (Tf), sample B: transferrin was 15% satured with 59Fe. Both Tf and Tf-bound 59Feactivity were eluted as bifid peaks; the smaller Tf peak eluted at fraction 64 was seen to coincide with the first peak of Tf-bound 59Fe.
TransfewinMolecular Diyerences
513
the molecular weight of transferrin to cqual 80 000. From the total iron content and TIBC, iron saturation of transferrin in each fraction was determined.
RESULTS The level of iron saturation of transferrin samples used in the experiments described below and the aniount of transferrin used on each colunxi is shown in Table I. Fig I shows the elution of iron-free transferrin from sample A which was eluted as a symmetrical peak betwcen fractions 60 and 80: the volume of eluted fractions was 3.5 ml. In four experiments, mean buffer conductance at peak protein concentration was 4.94+ 0.02 mmho at 250C. TABLE I. The level of iron saturation of transferrin in samples A-F and the amount of transferrin applied to each DEAE-cellulose column
I
Sample
A ~
B ~
:/, iron saturation mg transferrin/column
_
_
~
_
0
IS
21
40.4
IS
15
30
30
1
Sarnole C
E
D
C _
F
_ _ 58.5 30
_
_
_
I00
30
Sornple D
\
Tf -bound
/”Fe I
40
50
40
60
50
60
Froc tion
FIG 3. DEAE-cellulose chromatography of human transferrin (Tf).Iron saturation in samples C and D was 21% and 40.4% respectively. With increasing iron saturation of transferrin, the elution profile of Tf became more obviously bifid, the first eluted Tf peak increasing in size and being associated with the main peak of Tf-bound 59Feactivity.
_
R. S. Lane
514
Fig 2 (sample B) shows the optical density at 280 nm of the eluted transferrin (Tf) and the iron-transferrin complex (Tf-bound 59Fe)obtained from the 59Feactivity in each fraction. Fraction volume was 3.5 ml. Tf was eluted between fractions 60 and 80 and the profile was bifid; a small peak was seen at fraction 64 and the main peak at fraction 69. Tf-bound 59Fe also showed two peaks, the first at fraction 65 was slightly larger and coincided with the first peak of Tf; the second Tf-bound 59Fepeak was at fraction 74, that is, after the main Tf peak. Fig 3 shows the elution profiles of Tf and Tf-bound 59Fefrom samples C and D. In these experiments, the volume of each eluted fraction was 5.6 ml which accounts for the lower fraction numbers at which transferrin was eluted from D E A E - c ~ ~ ~columns. u~os~ Transferrin in sample C was 21% saturated with iron. Tf was eluted between fractions 40 and 60; peak concentration was at fraction 45 and a distinct shoulder to the main Tf peak was seen at fractions 42/43. As in sample B, Tf-bound 59Fefrom sample C showed two peaks, the first peak at fraction 43 was larger and coincided with the shoulder to the Tf peak; the second peak at fraction 49 was eluted after the main Tf peak. At the higher iron saturation of transferrin in sample C, the first eluted peak of Tf-bound 59Fe showed the relatively greater increase in size. Comparing the results of samples B and C, the greater definition of the Tf profile from sample B was obtained by the use of only 15 mg transferrin on the column and by collecting
Somple E
Fraction
FIG 4. DEAE-cellulose chromatography of human transferrin (Tf). Iron saturation in samples E and F was 58.5% and 100% respectively. In sample E, the first peak of Tf with its associated peak of Tfbound 59Feshowed that this sample contained more iron-complexed than iron-free transferrin. Sample F represented the elution of homogeneous Fe2-transferrin molecules which were eluted as a single homogeneous peak. In this sample the second peak of Tf-bound 59Fe was absent.
Transferrin Molecular DiJerences
51s
a smaller fraction volume. The use of 3 0 mg of transferrin and the collection of larger fractions in samples C-F, however, did allow more convenient manipulation of eluted samples. Transferrin in sample D was 40.4% saturated with iron. Tf was eluted in two peaks at fractions 43 and 47/48. Tf-bound 59Fe also showed two peaks: the first, at fraction 44, coincided with the first Tf peak, while the second, at fraction 51, characteristically followed the second peak of Tf. Compared with sample C, sample D contained twice as much iron. The bifid nature of the Tf peak was obvious and the association between the first peaks of Tf and Tf-bound 59Fereadily apparent. The increased amount of iron-transferrin complex in sample D presented mainly in the first Tf-bound 59Fepeak. Fig 4 shows the elution profiles of Tf and Tf-bound 59Fefrom samples E and F. Transferrin in sample E was 58.5% saturated with iron; there were two TEpeaks of which the first was larger and its association with the first peak of Tf-bound 59Feshowed that sample E contained more iron-complexed than iron-free transfcrrin. Equally important, the second peaks of Tf-bound 59Fein both samples D and E were approximately equal in size, thus this peak was maximal when initial iron-saturation in the transferrin sample approximated to 50%. In sample F, single peaks of Tf and Tf-bound 59Fewere seen together at fraction 43 and represented the elution of homogeneous Fez-transferrin molecules. In four experiments with iron-saturated transferrin, carried out in the manner used for apo-transferrin in sample A, peak elution of Fez-transferrin occurred at a mean level of buffer conductance of 4 . p + 0.01mmho at z5OC. DISCUSSION If iron binds with equal readiness at either binding site of transferrin (Aasa et al, 1963),then the proportions of apo-transferrin, Fe,-transferrin and Fez-transferrin in a transferrin solution of known saturation (S%) can be determined since the probability of an iron atom binding at any one site is S/IOO. It follows then: (100- S)2
Apo-transferrin Fe, -transferrin Fez-transferrin
I00
%
2s (100- S)
I00
%
S2 -% I00
Accordingly, the distribution of transferrin molecules in samples B-E are shown in Table 11. As the iron saturation in a transferrin sample increased from o to IOO%, the single peak of apo-transferrin was progressively replaced by two and then finally one peak of iron-transferrin complex. In the I 5 % iron-saturated sample B, Fez-transferrin molecules were negligible in number, yet two approximately equal peaks of iron-transferrin complex were eluted which strongly suggested that they contained equal populations of Fe,-transferrin molecules. As iron content in the transferrin samples increased so did their content of Fe,-transferrin molecules. Chromatographically, this became apparent as an increase in the first eluted peak
R. S. Lane
516
TABLE 11. The distribution of apo-transferrin, Fel-transferrin and Fez-transferrin molecules in samples B, C, D and E used for DEAE-cellulose chromatography Sample
Trangerrin iron-saturation
B C D E
% Apo-trangerrin
% Fel-transferrin
% Fe,-transferrin
molecules
molecules
molecules
72.25 62.4 35.5 17.2
25.5
15 21
40.4 58.5
2.25
33.2 48.2 48.6
4.4 16.3 34.2
TABLE 111. The level of iron-saturation and concentration of transferrin in selected fractions eluted during chromatography of samples C, D and E on DEAE-cellulose columns
Fraction
Sample C, 21%
iron-saturated
Sample D, 40.4%
iron-saturated
42 43 44 45 46 47 41 42 43 44 45 46 47 48 49 50
Sample E, 58.5%
iron-saturated
Transferrin (mglfracfion)
Iron saturation (%)
1.1
43 47 34
1.9 2.3 3.3 3.8 3.6 0.2
I2
6 3
2.3 2.6* 2.3
47 49 56 61 65
2.0
52
1.0
2.5
3.o 3.o 2.7
41 42 43 44 45 46 47 48 49
I .8 2.1
50
2.2
First Tf peak
'1 8
0.5
47
2.4 3.8* 3.7* 3.1 2.3 1.8
52
59 65 69 70 65 38231 16
Second Tf peak
First Tf peak Second Tf peak
* Fractions containing highest transferrin concentration within the first eluted peak of Tf in samples D and E.
Transfrrin Molecular Dtserences 517 of Tf-bound 59Fe which, in 100% iron-saturated transferrin, replaced both apo-transferrin and the second peak of Tf-bound 59Fe. Table I11 shows the levels of iron-saturation in transferrin fractions comprising the first and second peaks of Tf: within the first peak, fractions up to 43 were approximately 50% saturated and therefore these molecules were predominantly Fe, -transferrin. In samples D and E, fractions between 44 and 47 contained transferrin with levels of iron saturation approaching 70% and therefore these fractions contained a proportion of Fez-transferrin molecules. The distribution of iron saturation within the first Tf peak therefore showed that both Fe,-transferrin and Fez-transferrin molecules were present and that Fe,-transferrin was eluted first from DEAE-cellulose. In addition, Table I11 shows that the second Tf peak was mainly formed by apo-transferrin molecules. The iron status of transferrin molecules in the second peak of Tf-bound 59Fecould not be measured directly due to the proximity of the peak of apo-transferrin. However, other evidence indicated that this peak contained a second population of Fel-transferrin molecules : (I) Essentially, sample B was a mixture of apo-transferrin and Fel -transferrin molecules, yet two peaks of iron-transferrin complex were observed, of which the first was shown to contain Fe,-transferrin molecules. In sample B, both peaks of Tf-bound 59Fe were approximately the same in size, which would be expected in two populations of Fe,-transferrin molecules if iron atoms bind with equal facility at either metal-binding site of transferrin. (2) The maximum content of Fe, -transferrill molecules in transferrin samples occurs when net iron-saturation of the sample is 50%. Transferrin iron-saturation in sample D was 40.4% and in sample E was 58.5%, thus each sample contained equal proportions of Fe,-transferrin molecules. That the second peaks of Tf-bound 59Feeluted from samples D and E were equal in size, was consistent with these peaks containing Fe,-transferrin molecules. (3) In iron-saturated transferrin samples (Fez-transferrin), the second peak of Tf-bound 59Fewas absent: had this peak been attributable, not to Fel-transferrin, but to a more anodal transferrin variant or a proportion of partially denatured transferrin having a greater affinity for DEAE-cellulose than Fez-transferrin, then the second Tf-bound 9Fe peak would have persisted in the 100% iron-saturated transferrin sample. (4)It was assumed that iron in the first peak of iron-transferrin complex was attributable to half the sample population of Fe,-transferrin molecules and to the whole population of Fez-
TABLE IV. The amount of iron bound by transferrin in the first and second peaks of Tf-bound "Fe eluted from DEAE-cellulose columns. The first Tf-bound 59Fe peak contains both Fe,transferrin and Fe2-transferrin molecules. Iron bound by Fe,-transferrin within this peak is determined by subtraction and is shown to be equal to the iron bound by transferrin in the second Tf-bound 59Fepeak.
Sample
Iron saturation (%)
Total transferrinbound iron in -first peak (PA4
Iron infirst peak (pg)bound to Fe ,-trunsferrin
Iron in second peak ( p g ) of irontransferrin complex
B
IS
1.51
1.08
1.15
C
21
4.0
2.52
D
40.4
8.61
3.7
2.6 3.53
R.S . Lane
518
transferrin molecules; likewise iron in the second peak of iron-transferrin complex was attributed to a half-population of Fe,-transferrin molecules. Iron bound by Fe,-transferrin in each peak was determined and examples are given in Table IV. It is seen that equal amounts of iron were bound by Fe,-transferrin molecules in the two peaks of iron-transferrin complex, confirming the view that these peaks contained equal populations of Fe, -transferrin molecules. Fig 5 shows the relationship between chromatographic and the established electrophoretic behaviour of transferrin (Aisen et al, 1966) and the relative molecular charge of transferrin molecules. Electrophoretic behaviour correlates with net molecular charge whereas chromatographic behaviour does not : Fe, -transferrin and Fez-transferrin molecules, although more negatively charged, both behave as weaker anions than apo-transferrin on DEAEcellulose columns at pH 7.90; in addition, two forms of Fc,-transferrin molecule are identified by chromatography. For descriptive purposes, these molecules are termed Fe,-transferrin A and Fe,-transferrin B, where the former is eluted first from the column.
Increasing onionic affinity
- transferrin
DEAEcellulose: pH 7.90
Fe,
Electro phoresis : pH 8.40
Apo-transferrin
-
Fez transferrin
Apo - transferrin
Fe I - transferrin
Fe,
Fe, - transferrin
- tronsferrin
FIG 5. The relationship between the established electrophoretic behaviour of transferrin and its chromatographic behaviour on columns of DEAE-cellulose. The units of negative charge shown in Fet-transferrin and Fe2-transferrin molecules are relative to the molecular charge of apo-transferrin. Each unit of negative charge is due to the binding by transferrin of one bicarbonate ion with each atom of iron.
The physical properties of molecules which influence their electrophoretic mobility and affinity to anion-exchange media are different.The interaction between transferrin and DEAEcellulose is between reactive, charged, surface groups. This interaction i s complex, yet the equilibrium between buffer ions, protein, cellulose and DEAE groups is very susceptible to changes in charge, permitting sequential elution of molecules whose differences in reactive charge are small. Evidently, the surface reactive charge of Fe, -transferrin molecules is not uniform, half the molecular population exhibiting a significant decrease in electronegative characteristics. Such behaviour is best explained by relating it to the changes in molecular conformation that occurs in transferrin molecules when iron i s bound, Conformational change may be generalized, for example, affecting the hydrodynamic volume of transferrin (Charlwood, 1971) ; alternatively, there are changes localized in the region of the metal-binding sites, which Tan (1971) has suggested represent the major changes in conformation induced by iron binding. Charlwood has shown a stepwise re-
Transferrin Molecular Dixerences
519 duction in hydrodynamic volume of 0.7% in human transferrin after the binding of each iron atom and it seems improbable that this would permit chromatographic differentiation between two forms of Fe,-transferrin molecule. It is more likely that chromatographic behaviour of Fe,-transferrin molecules is caused by localized changes in conformation in the region of the binding sites. It is probable that the co-ordination of each metal-binding site by iron induces dissimilar changes in molecular conformation in the region of the sites: in the case of the binding site which combines with iron in Fe,-transferrin A, the resultant conformational change is associated with loss of a surface reactive charged group previously able to react with DEAE-cellulose. The exact explanation of these chromatographic results is not known due to lack of information about the molecular structure of transferrin. Decreased electronegative behaviour of Fel-transferrin A could result from an alteration in the reactivity of an a-carboxyl group which is negatively charged at pH 7.90. In support of this, under comparable experimental conditions, loss of a single charged sialic acid residue from transfcrrin causes a decrease in a&nity of transferrin for DEAE-cellulose similar to that caused by iron-binding in Fe,transferrin A (unpublished data). In addition, Bezkorovainy & Grohlich (1970) have provided evidence suggesting that reactivity of carboxyl groups is not the same in the region of both binding sites of human transferrin. The chromatographic data are consistent with other experimental evidence which shows differences between the metal-binding sites (Aisen et al, 1969; Aasa, 1972; Price & Gibson, 1972) and it is thought that the conditions of the chromatographic studies approach more nearly to the physiological environment than is often the case. The finding of two equal populations of Fel-transferrin molecules is in agreement with Aasa et a2 (1963) who described the binding sites of transferrin as independent and equivalent structures. It is of interest that in transferrin with iron saturation approaching the physiological range of 20-40%~ two populations of Fe,-transferrin molecules form the bulk of available irontransferrin complex. This could be entirely in accord with the views of Fletcher & Huehns (1968) that the properties of iron transport are not uniformly shared by all the metal-binding sites of transferrin. Could a difference in reactive charge associated with one metal-binding site have any influence 011 the process of iron transfer to cells? More than one factor is responsible for the release of iron from transferrin, but of interest is the fact that Bezkorovainy & Grohlich (1970) were able to chelate iron more readily from one binding site of transferrin after minimal chemical modification of reactive carboxyl groups on the molecule. In conclusion, co-ordination of the binding site in Fe,-transferrin A caused a significant reduction in affinity of transferrin for DEAE-celldose. Iron bound at the binding site in Fe,-transferrin B caused a minimal change in the chromatographic behaviour shown by apo-transferrin. Transferrin with iron bound at sites A and B (Fez-transferrin) behaved like Fe,-transferrin A on DEAE-cellulose columns, thus the charge effect associated with site A dominated the chromatographic behaviour of the molecule.
REFERENCES AASA, R. (1972) Reinterpretation of the electron paramagnetic resonance spectra of transferrins.
Biochemical and Biophysical Research Communications, 49s 806.
R.S. Lane AASA,R., MALMSTROM, B.G., SALTMAN, P.& VX”G ~ DT., (1963) The specific binding of iron(I1I) and copper(I1) to transferrin and conalbumin. Biochimica et Biophysica Acta, 75, 203. AISEN,P. (1974) The role of transferrin in iron transport. (Annotation). Britirh Journal of Haematology, 26, 159.
AISEN,P., AASA,R. & REDFIELD, A.G. (1969) The chromium, manganese, and cobalt complexes of transferrin. Journal qf Biological Chemistry, 2 4 , 4628.
AISEN,P., LEIBMAN, A. & REICH,H.A. (1966) Studies on the binding of iron to transferrin and conalbumin. Journal ofBiological Chemistry. 241, 1666. BEZKOROVAINY, A. & GROHLICH, D. (1970) Modification of carboxyl groups of transferrin. Biochimica et Biophysica Acta, 214,37. CHARLWOOD, P.A. (1971) Differential sedimentationvelocity and gel-filtration measurements on human
apotransferrin and iron-transferrin. Biochemical Journal, 125, 1019. FLETCHER, J. & HUEHNS,E.R. (1967) Significance of the binding of iron by transferrin. Nature, 215, 584. FLETCHER, J. & HUEHNS,E.R. (1968) Function of transferrin. Nature, 218, 1211. LANE,R.S. (1971) DEAE-cellulose chromatography of human transferrin: the effect of increasing iron saturation and copper(I1) binding. Biochimica et Biophysica Acta, 243, 193. PRICE,E.M. & GIBSON, J.F. (1972) Electron paramagnetic resonance evidence for a distinction between the two iron-binding sites in transferrin and in conalbumin. Journal of Biological Chemistry, 247, 8031.
TAN,A.-T. (1971) Circular dichroism properties of conalbumin and its iron and copper complexes. Canadian Journal of Biochemistry, 49, 1071.
