Chemical, but not functional, differences between the iron-binding sites of rabbit transferrin

Biochimica et Biophysica Acta, 701 (1982) 295-304

295

Elsevier Biomedical Press BBA31073

CHEMICAL, BUT NOT FUNCTIONAL, DIFFERENCES BETWEEN THE IRON-BINDING SITES OF RABBIT TRANSFERRIN T.A. DELANEY, W.H. MORGAN and E.H. MORGAN *

Department of Physiology, University of Western Australia, Nedlands, Western A ustrafia, 6009 (,4 ustralia) (Received June 17th, 1981)

Key words: Transferrin; Iron-binding site; (Rabbi 0

The interaction between iron and rabbit transferrin was investigated by three methods, (1) by studying iron release mediated by phosphatic compounds and iron chelators, (2) by determining the distribution of iron between the binding sites in the presence of chelators and (3) by measuring iron uptake from two iron-binding sites by rabbit reticulocytes. The distribution of iron between diferric transferrin and the two forms of monoferric transferrin was determined by electrophoresis in polyacrylamide gel containing 6 M urea. Iron release mediated by ATP, ADP, 2,3-diphosphoglycerate or citrate occurred at a single rate in a manner indicating that iron was released at the same rate from the two iron-binding sites and that iron release from either site was independent of that from the other. By contrast, iron release mediated by nitrflotriacetate, pyrophosphate or oxalate occurred at different rates from the two binding sites, more rapid release occurring from one site with nitrilotriacetate and pyrophosphate and from the other site with oxalate. The rates of iron release mediated by the above substances varied with incubation temperature and pH. In the presence of nitrilotriacetate, pyrophosphate or oxalate iron bound preferentially to that site from which it was more slowly released by the chelator. When transferrin was incubated with reticulocytes iron was taken up much more rapidly with diferric transferrin than from monoferric transferrin, but there was no evidence of preferential uptake of iron from either binding site. It is concluded that there are distinct chemical differences between the two iron-binding sites of rabbit transferrin. However, they were not reflected by functional differences under the conditions of incubation with reticulocytes used in this work. The results for iron release from transferrin mediated by the action of phosphatic compounds and chelators support the concept that protonation is involved in the rate-limiting step in the iron-release process.

Introduction The plasma iron-transport protein, transferrin, has two metal binding sites. There is abundant evidence for chemical differences between these sites in human and rat transferrins [1-5] but disagreement exists as to whether one of the sites is a * To whom correspondence should be addressed. Abbreviation: Hepes, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid. 016%4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

better donor of iron to cells than the other [1,6-8]. One expression of a difference between the sites is the rates at which iron is released in the presence of phosphatic compounds [9,10]. Another is the pH-dependence of iron binding to the sites [5,1113]. In contrast to human and rat transferrin there is no evidence of chemical differences between the iron-binding sites of rabbit transferrin [ 10,14-17]. Hence, it is possible that the reported differences in the rate of cellular uptake of iron from the two sites of human and rat transferrin and the failure

296

of earlier workers to find such a difference with rabbit transferrin [6-8] is a simple consequence of the presence or absence of chemical differences between the sites. However, van Baarlen et al. [18] have recently reported that one site of rabbit transferrin is a better donor of iron to rabbit reticulocytes than the other site, suggesting that there may be differences between the iron-binding Rites of the rabbit protein as with transferrins of other species. The aim of the present work was to investigate the process of iron release from rabbit transferrin in order to obtain further evidence as to whether the two iron-binding sites are chemically and functionally equivalent, and to provide more information on the mechanism of iron release from the protein. Three types of experiment were performed; viz. (1) iron release mediated by certain phosphatic compounds and iron chelators, (2) iron binding to transferrin in the presence of iron chelators and (3) iron release during uptake by rabbit reticulocytes. The distribution of iron between diferric transferrin and the two possible forms of monoferfic transferrin was determined by the use of polyacrylamide gel electrophoresis in the presence of 6M urea [19]. The results demonstrate chemical differences between the iron-binding sites of rabbit transferrin, but there was no evidence of differences in the rates of cellular uptake of iron from the sites. Materials and Methods

Chemicals. Hepes, ATP, ADP, 2,3-diphosphoglycerate, sodium citrate, sodium oxalate, nitrilotriacetic acid and sodium pyrophosphate were obtained from Sigma Chemical Co., St. Louis, MO, and desferrioxamine (desferrioxamine methane sulphate) from CIBA Ltd., Basle, Switzerland. Radioactive iron (FeC13, 10-30 #Ci/pg) and 1251 (NaI, carrier-free) were purchased from the Radiochemical Centre, Amersham, U.K. Purification and labelling of proteins. Transferrin was isolated from rabbit plasma in the diferric form by ion-exchange chromatography and gel filtration as in earlier work [10]. It was labelled with 1251 by the iodine monochioride method [20] while saturated with iron. Labelling with 59Fe was performed by first removing the iron from the

transferrin followed by addition of the radioactive iron in the form of iron-nitrilotriacetate, except where otherwise noted, and dialysis against 0.15 M NaC1. Radioactive iron was mixed with nonradioactive iron before labelling the protein so that 59Fe and carrier were added in a homogeneous form to achieve the desired degree of iron saturation. Reticulocytes. Reticulocyte-rich blood was obtained from rabbits which were bled 3-5 days after receiving five successive daily injections of phenylhydrazine in a dose of 6 mg/kg body weight. The cells were washed three times in ice-cold 0.15 M NaC1 before use.

Measurement of iron release from transferrin. The rate of iron release from transferrin in a cell-free system was measured as previously described [10] by measuring the change in absorbance at 295 nm of the difference spectrum between iron-transferrin and apotransferrin, using desferrioxamine as an acceptor of the released iron. Various mediators of iron release (phosphatic compounds and iron chelators) were added to the transferrin solution as indicated below. The solutions were buffered with 0.1 M Hepes. Absorbance measurements were made in a Varian Model 635 spectrophotometer fitted with a controlledtemperature cuvette holder. Except where indicated the conditions of incubation were as follows: temperature, 37°C; pH, 6.8-6.9; desferrioxamine concentration, 5 mM; mediator concentration, 5 mM; transferrin concentration 0.025 mM. Iron uptake by reticulocytes. Reticulocyte-rich rabbit blood cells suspended in Hanks and Wallace balanced salt solution [21] were incubated with rabbit transferrin labelled with 59Fe and t25I. After varying periods of incubation at 37°C sampies of the incubation mixture were removed, centrifuged at 4°C for 10 min at 1000 × g, aliquots of the supernatant taken for counting of radioactivity and analysis by urea-polyacrylamide gel eleetrophoresis and the cells washed three times in ice-cold 0.15 M NaC1 and counted for radioactivity. In certain experiments desferrioxamine was added to the incubation mixture at a concentration of 1.0 mM in order to prevent the possibility of any 59Fe or non-radioactive iron released from the cells being bound by the transferrin in the incubation solution. The reticulocyte count of the

297

cell suspensions varied from 40 to 70% and the haematocrit of the incubation mixture was 45-60%. Analytical methods. All glass and plastic was soaked in 0.1 M HC1, thoroughly rinsed with deionized distilled water and over-dried. Iron concentration was measured by the method advocated by the International Society of Haematology [22]. Protein was determined by a biuret method [23]. Reticulocytes were counted on dried smears after staining with new Methylene blue. Radioactivity was measured in a three-channel -/-scintillation spectrometer. The transferrin present in solution was separated into apotransferrin, monoferric transferrin and diferric transferrin by polyacrylamide gel electrophoresis in 6 M urea as described by Makey and Seal [19] using an LKB Multiphore electrophoresis apparatus. When the distribution of 59Fe between the different forms of transferrin was to be determined the samples were run in duplicate; one was fixed and stained for protein and the other sliced for counting radioactivity using a Hoefer gel slicer without fixing or staining. The distribution of protein between the different forms of transferrin was determined in one or both of two ways, viz. (1) slicing and counting for 125I and (2) staining and scanning the stained gels with a Gilford spectrophotometer fitted with a gelscanning attachment.

^

0"1

O05

It has been shown previously that incubation of diferric rabbit transferrin with desferrioxamine in the presence of ATP or 2,3-diphosphoglycerate results in the release of iron from the protein in a manner which has a single rate constant [10,16]. This was confirmed in the present work (Fig. 1A) and was also shown to occur when ADP or citrate were used as the mediator of iron release. However, when iron release was measured in the presence of nitrilotriacetate, oxalate or pyrophosphate, the curve for iron release could be resolved into two exponentials, each accounting for approx. 50% of the total iron present (Fig. 1B). These results suggested that iron release mediated by the latter three substances occurred at different rates from

0-05

40

80

120

160

Incubation

80

160

240

320

T i m e (rain)

Fig. l. Iron release from diferric rabbit transferrin mediated by 2,3-diphosphoglycerate (A) and nitrilotriacetate (B). The curve obtained with niirilotriacetate has been resolved into two exponentials. Incubation conditions as described in the test.

the two iron-binding sites, while iron release mediated by ATP, ADP, 2,3-diphosphoglycerate or citrate occurred at the same rate from the two

8

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6 A0o

Results Iron releasefrom transferrin mediated by phosphatic compounds and chelators

B

A

~4

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I

I

10

20

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Fig. 2. Distribution of 59Fe ( 0 O) and 125I (O .... O) between apotransferrin (Apo), A- and B-site monoferric transferrin (A, B) and diferric transferrin (Di) when a sample of rabbit transferrin labelled with SgFe and 125I was subjected to polyacrylamide gel dectrophoresis in the presence of 6 M urea and was then sliced and counted for radioactivity. The 59Fe and 125I counts were multiplied by ]0 - 2 and 1 0 - 3 respectively, to give the values shown on the figure.

298 sites. In order to test this hypothesis samples of rabbit transferrin were analysed by polyacrylamide gel electrophoresis in the presence of 6 M urea. This procedure has been shown to resolve human and rabbit transferrin and chicken conalbumin into apo- and diferric transferrin and two forms of monoferric transferrin in which the iron is situated in one or the other of the two binding sites [4,18,19,24]. Four protein bands were detected when rabbit transferrin, saturated to different degrees with iron, was analysed by urea gel electrophoresis. By varying the degree of saturation it was possible to demonstrate that the band closests to the origin corresponded to apotransferrin, the most distant to diferric transferrin and the intervening two bands to monoferric transferrin. This was confirmed by analysing samples of transferrin labelled with 59Fe and 12~I. The 59Fe: 125I ratios of the four protein bands corresponded to that expected from the above conclusion (Fig. 2). The two forms of monoferric transferrin will be referred to as A-site and B-site transferrin or A T and BT, the A-site form being the one closer to apotransferrin on urea-gel, electrophoresis. The relative distribution of transferrin between the four possible forms was quantitated by fixing and staining the gels and scanning for protein, or, when ~25I-transferfin was used, by slicing and counting the gel slices for radioactivity. Similarly, when 59Fe-labelled transferrin was used iron distribution was determined by slicing and counting for 59Fe. When samples of 1251-59Fe-labelled transferrin were taken at different times during the process of iron release mediated by 2,3-diphosphoglycerate and were analysed by urea gel electrophoresis it was found that the ratio of the distribution of 59Fe on the two forms of monoferric transferrin remained relatively constant. The amount of 59Fe on diferric transferrin decreased much more rapidly than did that on either form of monoferric transferrin, which did not start to decrease until the amount of 59Fe on diferric transferrin was approximately as low as that on the monoferric forms (Fig. 3A). When the total amount of transferrin-bound 59Fe and the amount on diferric transferrin were p l o t t e d semilogarithmically straight lines were obtained, the slope of the line for diferric transferrin-5gFe being twice as great as

A

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x

2

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I 50 Incubalion

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Time (mln)

Fig. 3. Distribution of 59Fe between diferric transferrin (A, A), A-site monoferric transferrin (¥, ~7) and B-site monoferric transferrin ( I , D) when a sample of rabbit transferrin labelled with t2sI and 59Fe was incubated at 37°C in the presence of 2,3-diphosphoglycerate (5 raM) and desferrioxamine(5 mM) at pH 6.8. The results in Fig. 3A are those obtained by counting the sliced gels for 59Fe (&, W, I ) and those calculated from the total 59Fe and the relative distribution of 125Ibetween the different forms of transferrin (A, ~, (~). Also shown are the theoretical values calculated using Eqns. I and 2 as described in the text (©). Fig. 3B is a semilogarithmicplot of the data for total 59Fe (Q) and S9Fe on diferric transferrin (A) during the incubation.

that for total transferrin-59Fe (Fig. 3B). This suggested that iron was being released at the same rates from the two sites of diferric transferrin and that iron release from either site was independent of that from the other, i.e., the rate constant of iron release is the same for the two sites and is the same whether the iron is released from diferric transferrin or from monoferric transferrin. If this is true, then,

where [AT]t is the concentration at time, t, of monoferric transferrin with iron at site A; [AT]i is its initial concentration; [ATB]i is the initial concentration of diferric transferrin; and k is the rate constant of iron release from transferrin. Also [ATB], = [ATBli e -2k,

(2)

Using the rate constant obtained from fig. 4B, and

299

A

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Fig. 4 Distribution of 59Fe between diferric transferrin (©), A-site monoferric transferrin (A) and B-site monoferric transferrin (&) when samples of rabbit transferrin labelled with SgFe were incubated at 37°C and pH 6.8 with 5 mM nitrilotriacetate (A), 5 mM oxalate (B) or I mM pyrophosphate (C), all in the presence of 5 mM desferrioxamine.

the values from [ATB]i and [AT]i obtained by slicing and counting the gels, it was possible to calculate the theoretical values of [ATB], and [AT]t. Similarly, by substituting B for A the values of [BT], were calculated. These theoretical results are shown in Fig. 3A. It can be seen that the experimentally derived points fit the theoretical ones closely. Similar results were obtained when ATP and citrate were substituted for 2,3-diphosphoglycerate in the type of experiment described above. In each case the data indicated that iron was released at similar rates from the two iron-binding sites and the rate constant for release was similar for diferric and monoferric transferrin. When nitrilotriacetate was used as the mediator of iron release the proportion of 59Fe on the two forms of monoferric transferrin changed markedly during the release process, indicating more rapid release of iron from the A-site than from the B-site (Fig. 4A). Differences in the rates of iron release from the two sites were also found when pyrophosphate or oxalate were used instead of nitrilotriacetate. With oxalate, release was more rapid from the B-site (Fig. 4B), but with pyrophosphate it was more rapid from the A-site (Fig. 4C). An additional study was performed with oxalate. A solution of diferric transferrin in which the anion at the iron-binding sites was oxalate rather than

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Fig. 5. Effect of pH on the rate constants of iron release from rabbit transferrin-iron-bicarbonate at 37°C mediated by a variety of iron chelators (5 raM) in the presence of desferrioxamine (5 raM). The chelators were nitrilotriacetate (A), oxalate (Q), 2,3-diphosphoglycerate ((3), citrate ([3) and ATP (A). Two sets of results are shown for nitrilotriacetate and oxalate, corresponding to the two rates of iron release found with these chelators. Also shown are the rate constants for iron release from the transferfin-iron-oxalate complex when 2,3diphosphoglycerate was used as the mediator (~7 . . . . . . ~7).

bicarbonate was prepared by a nitrogen purge method [25]. Iron release from this form of transferrin was then studied using 2,3-diphosphoglycerate as the mediator. By contrast to the results obtained with the transferrin-iron-bicarbonate complex the iron was released from the oxalate complex at two different rates, each accounting for approx. 50% of the total iron. The rate of iron release from transferrin mediated by all of the above substances varied with pH (Fig. 5) and temperature (Fig. 6). The rate increased as the pH was lowered in a direct linear relationship with the hydrogen ion concentration. The two rates of release from transferrin-iron-

300 citrate were used as the mediator. Two values for the activation energy were obtained with the other three mediators, corresponding to the two ironrelease rate constants. The values were 5.8 and 15.3 for nitrilotriacetate, 8.6 and 12.3 for oxalate and 13.4 and 18.6 k c a l / m o l for pyrophosphate.

Iron binding by rabbit transferrin

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, I 3.3

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Fig. 6. Effect of incubation temperature on the rate constants of iron release from rabbit transferrin-iron-bicarbonateat pH 6.8 mediated by several chelators (5 mM) in the presence of 5 mM desferrioxamine.The chelators were pyrophosphate (11), nitrilotriacetate (4,), oxalate (0), 2,3-diphosphoglycerate(O), citrate (D) and ATP (/x). Two sets of results are given for pyrophosphate, nitrilotriacetate and oxalate corresponding to the two rates of iron release observed with these chelators.

oxalate which were obtained using diphosphoglycerate as the mediator were much less sensitive to changes in p H than was iron release from the transferrin-iron-bicarbonate complex (Fig. 5). The activation energies of the release rates calculated from the Arrhenius relationship between the logarithm of the rate constant and the reciprocal of the absolute temperature were found to be 15-16 k c a l / m o l when ATP, 2,3-diphosphoglycerate and

The above results demonstrated differences between the iron-binding sites of rabbit transferrin which were manifest by the rate of iron release mediated by certain compounds but not by others. It was thi~refore of interest to determine whether or not iron added to transferrin in the form of complexes of these compounds was bound equally to the two sites. Iron was added to apotransferrin dissolved in 0.1 M NaCI/0.05 M Hepes (pH 7.4)/0.02 M N a H C O 3 in an amount sufficient to achieve 50% saturation of its iron-binding capacity. The iron : chelator concentration ratio was 1 : 4. The mixtures were allowed to stand at 23°C for 1 h and were then analysed by urea-polyacrylamide gel electrophoresis. The results of the experiment are summarized in Table IA. When the iron was added in the form of its complex with nitrilotriacetate, citrate or pyrophosphate, relatively more of the iron was bound to site B than to site A. However, iron as the oxalate complex bound preferentially to site A, rather than to site B. In a second experiment equimolar amounts of apotransferrin and diferric were mixed in the presence of the above substances at concentrations of 5 raM, except for pyrophosphate, 0.25 mM, at p H 7.4 (0.05 M Hepes/0.1 M NaC1/0.02 M N a H C O 3) and allowed to stand at room temperature for 16 h. The samples were then analysed by urea gel electrophoresis. In the absence of the chelators there was no redistribution of iron between the two forms of transferrin. However, when chelators were present iron was exchanged between the diferric transferrin and apotransferrin, so that after 16 h the iron was predominantly on the B-site when nitrilotriacetate, citrate or pyrophosphate were present and mainly on the A-site when oxalate was present (Table IB).

Iron release from transferrin mediated by reticulocytes Samples of reticulocytes were incubated with

301

TABLE I DISTRIBUTION OF TRANSFERRIN BETWEEN APOTRANSFERRIN, A-SITE AND B-SITE MONOFERRIC TRANSFERRIN AND DIFERRIC TRANSFERRIN In Experiment A, iron was added to apotransferrin to 50% saturation in the form of complexes with the chelators indicated. The molar ratio of iron:chelator was I :4. Urea gel electrophoresis was performed I h after adding the iron and protein distribution was determined by gel scanning. In Experiment B, apotransferrin and diferric transferrin were mixed, then the chelators were added to a concentration of 5 mM, except for pyrophosphate which was at 0.25 mM. Electrophoresis was performed after standing for 16 h at room temperature and protein distribution was determined by gel scanning. Chelator

Transferrin (% total) Monoferric transferrin

Apotransferrin

Experiment A Citrate Nitrilotriacetate Pyrophosphate Oxalate Experiment B Nil Citrate Nitrilotriacetate Pyrophosphate Oxalate

Diferric transferrin

A-site

B-site

28 21 28 24

12 22 22 29

34 38 28 22

26 19 22 25

48 27 22 24 28

3 18 15 17 33

4 31 47 37 9

45 24 16 22 30

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Fig. 7. Distribution of SgFe between diferric transferrin (0, ©) and A-site (11, D) and B-site (&, A) monoferric transferrin when rabbit transferrin labelled with 59Fe and t25I was incubated with rabbit reticulocytes. At the indicated times samples of the incubation solution were analysed by urea-gel

59Fe-12SI-labelled transferrin for varying periods of time. Radioactivity was then measured in the washed cells and the incubation solution, and aliquots of that solution were fractionated by ureagel electrophoresis. Fig. 7 illustrates the results obtained in one experiment. Radioactive iron disappeared from the incubation solution and appeared in the cells. Disappearance from the incubation solution was most rapid from diferric transferrin and less rapid from the two forms of monoferric transferrin from which it disappeared at rates which were approximately proportional to their relative concentrations. Almost the same distribution of 59Fe between diferric and the two forms of monoferric transferrin was obtained by direct measurement of 59Fe in the sliced gels or by calculation from the total 59Fe in the incubation solution and the relative distribution of 1251 beelectrophoresis. The figure shows the values obtained by counting the sliced gels for 59Fe (O, II, A) and those calculated from the total ~gFe applied to the gels and ~2sI values of the slices. The initial concentration of diferric transferrin was 4.5 ~M.

302 tween the three forms of iron-transferrin (Fig. 7). Seven experiments of the type illustrated in Fig. 7 were performed, using different samples of reticulocytes and samples of transferrin with varying degrees of iron saturation present at different concentrations. A similar pattern of results was obtained in all of the experiments. This pattern was that iron was released from diferric transferrin much more rapidly than from the monoferric transferrins. The amounts of 59Fe on monoferric transferrin remained almost constant until the concentration of diferric transferrin had fallen to between 1.3 and 2.6 #M. There was no evidence from any of these experiments of preferential cellular uptake of iron from one of the two ironbinding sites of tansferrin. Desferrioxamine was added to the incubation solution in two of the experiments at a concentration of 1 mM, which has been shown not to cause a decrease in iron uptake by reticulocytes [26]. No difference in distribution of 59Fe between diferric and the monoferric transferrins was found between these samples and samples from other incubations performed under the same conditions except that desferrioxamine was not present. Discussion The present investigation confirms the observation [18] :that rabbit transferrin, like human transferrin and conalbumin, can be separated into apotransferrin, two forms of monoferric transferfin and diferric transferrin by polyacrylamide gel electrophoresis in the presence of 6 M urea. In the case of human transferrin and conalbumin, the form of monoferric transferrin with the slower electrophoretic mobility has its iron at the binding site which is situated in the C-terminal part of the molecule and that with the faster mobility binds the iron in the N-terminal part [24]. The same may be true of rabbit transferrin so that the A-site may be in the C-terminal and the B-site in the Nterminal parts of the molecule. However, this has not yet been proven and the A and B-site terminology is therefore used in this paper. The results for iron release mediated with nitrilotriacetate, pyrophosphate and oxalate and for iron binding in the presence of these substances provides evidence that there are distinct chemical

differences between the two iron-binding sites of rabbit transferrin. These differences are not manifest by the pH-dependence of iron binding to the sites [17] or by the present results for iron release mediated by ATP, ADP, 2,3-diphosphoglycerate or citrate. Both nitrilotriacetate and oxalate are iron chelators which can replace bicarbonate (or carbonate) as the anion present in the iron-binding site as part of the transferrin-iron-anion ternary complex [25]. Possibly the differences between the two binding sites of rabbit transferrin are primarily reflected in differences in the anion-binding part of the site. These differences have little effect when bicarbonate is the anion, but nitrilotriacetate appears to bind more readily to the B-site and oxalate to the A-site of transferrin than to the opposite site in each case. The result is preferential binding of iron to the appropriate site and slower release from the same site in the presence of one or other of these two anions. This suggestion is supported by the results obtained for iron release from the transferrin-iron-oxalate complex when 2,3-diphosphoglycerate was used as the mediator. • Two rates of iron release were observed, probably because the iron oxalfite bound more strongly to one site than to the other. It has been shown previously that there is a direct linear relationship between the H + concentration and the rate of iron release from human, rat and rabbit transferrin [9,10,27]. The present results extend these observations on rabbit transferrin to a side range of mediators of iron release. On the basis of the earlier work, it was proposed that iron release from transferrin involves an initial interaction between H + and the iron-transferrin complex followed by release of iron under the action of the mediator [10]. The present results are compatible with this hypothesis. The observation that the activation energy of iron release from both sites when ATP, diphosphoglycerate or citrate were used as the mediators or from one site when nitrilotriacetate was used were similar suggests that the same type of mechanism is operative in each case. Presumably, this could be largely determined by the energy required for the interaction of protons with the site in such a way as to weaken the complex between the iron and transferrin so that it can be bound by the mediator and transported from the iron-binding site. The

303 higher activation energy levels for iron release from the second, slower releasing site of transferrin in the presence of nitrilotriacetate, pyrophosphate or oxalate indicates that a higher energy barrier must be overcome to allow iron release, possibly because the complex of iron with the mediator can bind to the iron-binding site of transferrin. The rates of iron release from the two sites of rabbit transferrin fitted very closely to the theoretical values calculated on the basis of the model presented in the Results section of this paper. This model assumed that iron release from one site occurred independently of iron release from the other and that when ATP, ADP, 2,3diphosphoglycerate and citrate were used as the mediator, the iron was released at equal rates from the two sites. Hence, it may be concluded that these assumptions are correct, at least to the level of sensitivity allowed by the experimental methods which were employed. The differences in the iron-binding properties of the two sites of rabbit transferrin were not expressed in the rates of iron uptake by reticulocytes from the sites. However, the results did show evidence of a markedly greater rate of iron release from diferric transferrin than from the two forms of monoferric transferrin. The most likely explanation for the effect is that the affinity of the cell membrane receptors is higher for diferric transferrin than for monoferric transferrin and that in most instances when diferric transferrin is taken up by the cell both of its iron atoms are assimilated by the cell before the protein moiety of transferrin is released back to the incubation medium. Hence in most instances the observable sequence in experiments suh as the present ones is diferric transferrin--, apotransferrin, rather than diferric transferrin --, monoferric transferrin --, apotransferrin, which occurs in the cell-free system. The suggestion that the affinity of the receptors is greater for diferric transferrin than for monoferric transferrin is supported by the observations that the concentration in the incubation medium of the monoferric transferrins did not commence falling until the concentration of diferric transferfin was between 1.3-2.5 #M. The transferrin receptors on rabbit reticulocytes become saturated

with diferric transferrin when it is present at about this concentration. Presumably at lower concentrations not all of the receptors are occupied by diferric transferrin and are therefore available for reaction with monoferric transferrin, for which they have a lower affinity. An alternative explanation to the above is that there is little or no difference between the affinities of the cellular receptors for diferric transferrin and monoferric transferrin, and that in most instances diferric transferrin gives up only one of its iron atoms before being released from the cell. This would lead to a more rapid decrease in the concentration of diferric transferrin in the incubation medium than of monoferric transferrin, as was observed with the cell-free iron-release system. However, the concentrations of the monoferric species would have increased much more than was observed (Fig. 7). Hence the former explanation appears to be more likely than the latter. One concern with the experiments involving incubation of labelled transferrin with reticulocytes was that any iron contamination of the incubation solutions or test tubes, or the possible release of iron from the cells, could lead to false results by binding to transferrin in the solution, converting apotransferrin to monoferric transferfin and monoferric transferrin to diferric transferfin. However, two lines of evidence indicate that this was not a problem. Firstly, almost identical results for iron release from the two iron-binding sites were obtained whether they were based on direct measurement of 59Fe in gel slices, or on the relative proportion of transferrin between its possible forms as determined by counting gel slices for 125I (Fig. 7). This would not have been so if contamination was occurring because the binding of contaminating non-radioactive iron to the transferrin would have led to differences in the distribution of radioactive iron between the different forms of transferrin as determined by the two methods. The second type of evidence is derived from the results obtained when desferrioxamine was present in the incubation medium. This chelator was present at more than 50-times the molar concentration of transferrin. Hence it should effectively bind any contaminating iron. However, the presence of desferrioxamine did not result in different results for iroin distribution between the

304 forms of transferrin t h a n those f o u n d in its absence. The present results are c o n t r a r y to those of van Baarlen et al. [18], who reported evidence that the B-site of r a b b i t transferrin is a better d o n o r of iron to rabbit reticulocytes than is the A-site. They separated the different forms of the transferrin by gel electrophoresis b u t q u a n t i t a t e d them by a crossed i m m u n o e l e c y r o p h o r e t i c method. The reason for the conflicting results is uncertain. Possibly they are due to differences in the analytical methods which were used. Alternatively, the observation of functional heterogeneity of the i r o n - b i n d i n g sites of r a b b i t transferrin u n d e r some circumstances a n d not in others m a y be a result of variation i n cellular function. F u r t h e r work is required to answer these questions.

Acknowledgements T h e writers are i n d e b t e d to Miss O. Siira for skilled technical assistance. The work was supported b y research grants from the A u s t r a l i a n Research G r a n t s C o m m i t t e e a n d the University of W e s t e r n Australia. T.D. was the recipient of a R a i n e Vacation Scholarship.

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