The effect of pH on the kinetics of iron release from human transferrin

140

Biochimica et Biophvsica Acta, 719 (1982) 140 146 Elsevier Biomedical Press

BBA 21235

T H E E F F E C T O F pH O N T H E K I N E T I C S OF I R O N RELEASE F R O M H U M A N TRANSFERRIN D A V I D A. B A L D W I N , D E O L 1 N D A M.R. DE SOUSA and RA1NER M.A. VON W A N D R U S Z K A

Department of Chemistry, University of the Witwatersrand, 1 Jan Smuts Avenue, Johannesburg 2001 (Republic" of South Africa) (Received February 15th, 1982) (Revised manuscript received June l lth, 1982)

Key words: Transferrin; pH effect," Iron release kinetics," (Human)

The rate of iron release from the N-terminal and C-terminal monoferrictransferrins (FeN-transferrin and Fec-transferrin , respectively) has been studied at 37°C over the pH range 3.5-10.6 using EDTA as the accepting chelate. FeN-transferrin is the more facile except above pH 8.2. Plots of log10 kobs against pH showed a deviation for both monoferrictransferrins between pH 5.6 and 6.0 and studies above and below this transition point indicated that iron release occurs by different mechanisms. At low pH (<5.6) the rate of release from FeN-transferrin is independent of the presence of EDTA or NaCIO4, whereas Fec-transferrin shows a small but significant increase with increasing EDTA concentration. Rapid protonation of both monoferrictransferrins is followed by relatively slow release of Fe 3+ which is subsequently chelated by EDTA. The slower release from Fe c-transferrin is probably due to its greater binding strength for iron and the greater conformational stability of the C-terminal domain. Above pH 6.0 iron release from both monoferrictransferrins increases as the concentration of EDTA is increased. Direct attack of EDTA probably occurs giving Fe-transferrin (HCO~)- EDTA as a transition state or intermediate. The factors which may lead to the observed pH dependence of the rate include (i) protonation of groups directly bound to the iron, (ii) conformers which differ in degree of protonation and (iii) the degree of protonation of the attacking chelating agent. It is suggested that an increase in conformational fluctuations as the pH is lowered may play a very important role. Studies with diferrictransferrin at pH 4.53 and 7.40 showed that when iron is released to EDTA the rate is independent of the occupancy of the other site; that is, the two sites are exhibiting non-co-operativity.

Introduction H u m a n transferrin binds two Fe 3 + ions at sites located near the C-terminal and N-terminal regions of the protein [1,2]. The two iron-binding sites show a number of differences in vitro, for example, their binding strengths for Fe 3+ [3,4] spectroscopic properties [3], their accessibility to various iron complexes [3] and the rate at which iron is released to chelating agents [5-7]. However, one of the most important differences is the effect of p H on the relative iron binding strengths. The 0 3 0 4 / 4 1 6 5 / 8 2 / 0 0 0 0 - 0 0 0 0 / $ 0 2 . 7 5 © 1982 Elsevier Biomedical Press

N-terminal site does not bind iron below pH 5.7 [8,9] whereas the C-terminal site remains occupied in the absence of chelating agents down to p H 4.8 [8]. The weaker binding strength for iron at low p H has been utilized for many years for the preparation of apotransferrin and, more recently, the selective binding at the C-terminal site between p H 5.0 and 6.0 has been exploited to prepare the C-terminal monoferrictransferrin, Fe¢,-transferrin [6,10] and to selectively label the two sites with radioactively labelled iron [10]. When Fe 3+ binds to apotransferrin three protons are released for

141 each iron bound [11]. The source of these protons has been generally attributed to the binding of three tyrosine residues [11,12], although recent evidence suggests that only two tyrosines bind and that the third proton is released from the water molecule bound to the iron [12,13]. The relative affinity of iron for the two sites and the co-operativity of binding have been shown to depend on the ionization of functional groups of p K a approx. 7.4 (possibly histidines) [4]. However, salts such as NaC1 can affect both the relative binding strength of the two sites, i.e. the thermodynamics [4] and the relative rate of iron release to chelating agents, i.e. the kinetics [5,6], around physiological pH. The effect of p H on the rate of iron release from the two sites is of considerable interest since protonation must occur during the iron release reaction. Protonation of the synergistic anion, bicarbonate, and of groups on the protein have been proposed as important steps during iron release [1]. Also, Morgan [2] has suggested that iron release may occur within endocytotic vesicles into which protons are transported by an energy-dependent process. Although the rate of iron release from diferrictransferrin, Fe2-transferrin (or Fe c- or FeN-transferrin ) is known to increase dramatically on lowering the p H [14] no study has compared the effect of p H on all three ferrictransferrins. With the ready availability of kinetically pure samples of the two monoferrictransferrins [6], we have extended our previously reported studies on iron release to E D T A at p H 7.4 [5,6] to the p H range 3.5-10.6. Methods Diferrictransferrin and the two monoferrictransferrins, prepared as previously described [6], were dialyzed against Tris-HC1 buffer, p H 7.4 of low ionic strength ( I = 0.01) and diluted to A466 of 0.300 and 0.150, respectively. Fast kinetics (t,2< 45 s) were studied using a Durrum D110 Stoppedflow Spectrometer: the data were collected on a Datalab DE901 transient recorder which was interfaced to a 48K Apple II Plus Microcomputer. The curves were analyzed by the computer using the normal semilog plots with a minimum of 200 data points. The plots which were linear over at least 4 half-lives were subjected to least-squares

least-squares analysis; the reported rate constant being the average of at least three runs. One drive syringe contained the ferrictransferrin solution described above and the other an E D T A solution in a buffer ( I = 0.1) at an appropriate p H (see below). Slow kinetics were followed as previously described [5,6] except that 1 ml of the ferrictransferrin was added to 1 ml of EDTA in buffer ( I = 0.1). E D T A solutions were prepared by dissolving the appropriate amount of the disodium salt in about 50 ml of the buffer ( I = 0 . 1 ; acetate, p H 3.6-5.8; phosphate, p H 5.5-8.5; Tris-HC1, pH 7.0-10.5) and adjusting the p H using the appropriate base or acid. The solution was then made up to 100 ml with further buffer. Mixing equal volumes of an E D T A solution ( I = 0 . 1 0 ) and a ferrictransferrin solution ( I = 0.01, p H 7.4) altered the p H of the former by less than 0.1 pH units. The reported p H values have, however, been corrected for this change. Studies in the regions where the buffer ranges overlapped showed that within experimental error there was no influence of buffer on the observed rate constants. The addition of excess apotransferrin also had no effect on the rate of reaction. Unless stated otherwise complete removal of iron was observed at all times. Results In this study we have emphasized the iron release reaction from the two monoferrictransferrins since excellent first-order curves were obtained. Iron release from Fe2-transferrin normally gives multiphasic kinetics which necessitates the use of curve stripping procedures and results in less accurate values for the rate constants [5]. The rate constants (kobs) obtained when iron is removed from the two monoferrictransferrins in 10 m M E D T A from p H 3.5 to 10.6 are shown graphically in Fig. 1 and a selection of the data is given in Table I. Iron release from FeN-transferrin is more facile except above p H 8.2 where iron release becomes extremely slow (t,2 approx. 21 h, p H 8.6). No iron loss could be detected from FeN-transferrin between p H 9 and 10 over a period of 72 h nor was there any change in the visible spectrum. The two monoferrictransferrins are thus not identical above p H 9.0 as might be inferred from a previous

142 TABLE I V A R I A T I O N OF koB s F O R F e c - T R A N S F E R R I N F e N - T R A N S F E R R I N W I T H p H ([EDTA]--10 mM)

AND

N,s., no signifcant loss of iron observed over a period of 72 h. Fe N-transferrin

Fec-transferrin

pH

pH

kob s (rain t)

kob s (min 1)

3.51 4.02 4.58 5.02 5.55 6.05

4.34.103 7.06.102 1.20.102 3.18.101 4.98.10 ° 3.24.10 °

3.80 4.04 4.52 5.03 5.58 6.10

1.94.102 3.05- 10 I 1.56.10 ° 4.54- 10 i 1.74.10 l 1.42.10 " I

6.63 7.13 7.38

3.04.10- l 4.79.10 2 2.14.10 2

6.64 6.95 7.41

2.59.10 1.06.10 4.73.10

2 6 3

7.90 8.60 9.65 10.40

4.00.10 3 5.48.10 - 4 n.s. n.s.

7.95 8.50 9.65 10.40

1.73.10 9.72- 10 6.48- 10 5.68.10

3 4 4 4

report [15]. The maximum difference in rate of release occurs at pH 4.7 when iron release from Fey-transferrin is about 90 times faster than from Fec-transferrin. The major feature of the curves shown in Fig. 1 is the appearance of a transition

4

3 \:\ 2

\\

log kobs 0 ( rnlr~ll

*\

X

*".,.

"K'**t*i

-1

point between pH 5.6 and 6.0, which together with evidence to be presented below suggests that at this pH a major change occurs in the kinetic behaviour of both monoferrictransferrins. The kinetics above and below this transition point will be discussed separately. Low p H ( < 5.6) The kinetic behaviour of FeN-transferrin in this pH region is fairly straightforward; the plot of log kobs against pH is linear with a slope of - 1.41 +0.03 (Fig. 1) which suggests the involvement of more than one proton. Studies at pH 4.53 showed that the rate is completely independent of the concentration of EDTA (0-50 mM, kob s = 130-+ 10 min - l ) and of the presence of NaC104 (0-50 m M , k o b s = 126-*-4 min-I). The kinetics for Fec-transferrin are somewhat more complex. The plot of log kobs against pH shows a pronounced curvature (Fig 1) and the rate constants at pH 4.53 have a small but definite dependence on the concentrations of EDTA and NaC104 (Table II). The effect observed with the latter is not simply due to an increase in ionic strength since the increase in rate is greater than that observed with EDTA which provides a greater ionic strength contribution ( p r e d o m i n a n t l y E D T A H ~ - at pH 4.53). The NaC104 may be aiding iron release from Fec-transferrin by promoting a conformational change as has been observed at pH 7.4 [7]. In the absence of EDTA at pH 4.53, Fec-transferrin loses about 50% of its iron fairly rapidly (t, approx. 8 min); this step is then followed by a much slower loss of iron. The above results suggest that at low pH iron release from both monoferrictransferrins ocurs by the following mechanism (Tf, transferrin):-

~',

K

~¢\X

F e T f ( H C O 3 ) + n H + ~ F e T f ( H C O 3 ) H,~ +

\,

\ •be

Jr

kl

F e T f ( H C O 3 ) H ,"+ ~ -

3

k

\~-'t,~,.,,

. . . .

,,Fe 3 + , , + E D T A 4 -,~

4

5

6

7

_

9

10

(l)

"Fe 3 + ' ' + T f H ~ - I 3 ) + + H 2 0 + C O 2 ( 2 ) 1

k2

~[FeEDTAH20 ]-

(3)

11

pH

Fig. I. Plot of log k o b s for Fey-transferrin × × and (C)Fe-transferrin ( 0 O) against pH ([EDTA]= 10 mM).

In steps 2 and 3, "Fe 3+ " denotes the hydroxyferric complexes that are known to occur at this pH. The proposed mechanism involves rapid pro-

143 T A B L E II E F F E C T O F EDTA A N D NaCIO 4 C O N C E N T R A T I O N S ON kob s F O R F e c - T R A N S F E R R I N AT pH 4.53 [EDTA] (mM) [C104 ] (mM) kob s (min -1 )

2.5 0 1.14

5.0 0 1.32

10.0 0 1.56

15.0 0 1.74

tonation (Step 1), a relatively slow conformational change that results in release of Fe 3+ to the bulk solvent (Step 2) followed by chelation of the Fe 3 + by EDTA (Step 3). The difference in rates observed for the two monoferrictransferrins is primarily due to the values of k t and k_ Z, that is their relative conformational stability. For Fey-transferrin, the complete loss of Fe(III) in the absence of EDTA and the independence of the rate on EDTA concentration indicates that k t must be much greater than both k_ t and k 2. The rate of iron release from Fey-transferrin is thus only determined by k I and the size of the equilibrium constant, K. Fec-transferrin, on the other hand, only loses 50% of its iron rapidly in the absence of EDTA which suggests that k t and k_ t are approximately equal. The subsequent very slow loss of iron in the absence of EDTA is probably due to slow aggregation of 'Fe 3+ ' and loss of CO 2 to the atmosphere which will swing the equilibrium (step 2) to the right. In the presence of EDTA the 'Fe 3+' will be removed by step 3 and the equilibrium will shift in favour of apotransferrin and [ F e E D T A H 2 0 ] - . The rate of iron release from Fec-transferrin should increase to a maximum value as the EDTA concentration increases. That this is correct is suggested by the results in Fig. 2 although a plot of 1/kob s against 1/[EDTA] is non-linear which indicates that the actual mechanism may be more complex. The slower rate of iron release from Fec-transferrin compared to Fey-transferrin is consistent with the expected lower conformational stability of the N-terminal site. In the proposed mechanism only step 1 involves direct protonation, however protons will be released on formation of the ferrichydroxycomplexes in step 2 and on formation of [ F e E D T A H 2 0 ]- in step 3 since the EDTA is present predominantly as E D T A 2 below pH 6.00 [16]. Fey-transferrin

25.0 0 1.92

50.0 0 2.16

10.0 10.0 2.58

10.0 25.0 3.78

10.0 50.0 5.04

which is independent of the EDTA concentration would thus be expected to show a simpler dependence on pH than Fec-transferrin, as is observed. At pH 4.53, diferrictransferrin gives a fast phase, kobs = 125 min l, and a slow phase, kobs z 1.62 min i, in 10 mM EDTA. These rates are similar to those obtained for Fey-transferrin and Fe ctransferrin, respectively (see Table I). Since the fast phase should represent loss of iron predominantly from the N-terminal site of Fez-transferrin [5] this result suggests that the rate is independent of the occupancy of the C-terminal site.

High p H ( > 6. O) For FeN-transferrin, the plot of log kobS against p H is linear between pH 6.0 and 8.0 (Fig. 1) with a slope of --1.62---0.04 again suggesting the involvement of more than one proton. However, in contrast to the results at low pH, the rate is dependent on the concentration of EDTA (Table III). Also we have previously shown [6] that lyo-

2.(3

J J"

kobs(minI)

0

/

/

I

10

2;0

I

3;0 40 [EDTA], mM

5;0

Fig. 2. Plot of kob s ( m i n - I ) for Fec-transferrin against the concentration of EDTA, m M at pH 4.53.

144 TABLE III V A R I A T I O N OF kob s WITH EDTA C O N C E N T R A T I O N AT pH 7.40 (TRIS-HCk I =0.05) Values represent 102 kob ~, min

i.

[EDTA] (mM)

Fe Ntransferrin

Fe ctransferrin

Fe:-transferrin ~'

5 l0 25 50 100

1.62 2.01 2.32 2.48 2.70

0.28 0.47 0.77 1.10 1.73

1.89 2.40 3.00 3.49 4.46

0.27 0.43 0.87 1.19 1.72

a Data taken from Ref. 5.

tropic anions such as C104 decrease the rate of iron release from FeN-transferrin at pH 7.4 whereas cations such as Li + give an increase. Fec-transferrin again shows more complex behaviour than FeN-transferrin; the log kob s v e r s u s p H plot (Fig. 1) showing a pronounced curvature. The rate of iron release is also dependent on the E D T A concentration (Table III). Previous studies at p H 7.4 have shown that Fec-transferrin undergoes a conformational change in the presence of the lyotropic agents. NaCIO 4 and LiC1 which results in an increased rate of iron release [6]. From these results it seems reasonable to conclude that iron release from both monoferrictransferrins around physiological p H requires the attack of the negatively charged EDTA. A possible intermediate is a "quarternary" complex of the type, Fe-transferrin (HCO3). EDTA as has been proposed by Bates [17]. It should be emphasized, however, that no spectral intermediate has been observed during iron release [5]. Iron release from Fe2-transferrin at pH 7.4 has been studied previously [5] and these data are included in Table III. Two distinct phases are observed with Fe2-transferrin and comparison of the rates with those of the monoferrictransferrins shows that the slow phase corresponds to iron release from Fec-transferrin. Theoretically the fast phase should equal the sum of the rate constants for iron release from the N-terminal and C-terminal sites of Fe2-transferrin; that is when the other site is occupied [5]. From Table III, it can be seen that the fast phase is, within experimental error,

equal to the sum of the rates observed for the individual monoferrictransferrins. Thus iron release from Fe2-transferrin under these conditions occurs from two essentially independent sites; the rate not being influenced by the occupancy of the other site. Although only two phases are observed with Fe2-transferrin a third intermediate phase corresponding to iron release from FeN-transferrin should be present. The percentage change in absorbance expected for this phase would however be small because of the relative values of the rate constants (Table III) and the lower molar extinction coefficient of FeN-transferrin compared to Fec-transferrin [ 18]. Discussion

Although the two monoferrictransferrins release iron at significantly different rates throughout almost the whole of the pH range studied, (3.5 10.6), a change in mechanism occurs for both species in the p H range 5.6-6.0. Above p H 6.0 iron release from both monoferrictransferrins requires the direct attack of the accepting chelate E D T A and the probable formation of a "quarternary" complex, Fe-transferrin(HCO3). EDTA. A similar mechanism has been postulated for other chelates in this p H range [17]. At pH values below the transition point the rate of iron release from both species depends primarily on the rate of unfolding of the protein, the iron being released to the solution and chelated by the EDTA. The difference in observed rate between the two monoferric-transferrins is apparently due to the greater conformational stability of the C-terminal domain. Studies at both p H 4.53 and 7.40 suggest that the rate of iron release from either site is independent of the occupancy of the other, that is they exhibit nonco-operativity at least under these conditions. Both above and below the transition point the variation of kob~ with p H does not result in a simple sigmoid dependence for either of the monoferrictransferrins. Even though the linear dependence of the log k o b s v e r s u s pH plot for FeN-transferrin suggests that about 1.5 protons are involved there must be more than one factor which is operative. These most likely include (i) protonation of groups directly bound to the iron, e.g. the bicarbonate (or carbonate) synergistic anion,

145 O H , etc., (ii) conformers which differ in theiJ degree of protonation and (iii) the degree of protonation of the attacking chelate. Direct protonation of the bicarbonate (or carbonate) synergistic anion prior to iron release has been proposed as a required step in the reaction [1,19,20]. Subsequent loss of the anion either a s HzCO 3 or possibly as CO 2 as has been observed with model carbonate complexes [21,22] would then result in a relatively unstable ferrictransferrin complex, possibly Fe-transferrin. E D T A around physiological p H and Fe-transferrin below the transition point (pH < 5.6). Although an increase in rate of iron release at low p H is suggested by a mechanism of this type the actual dependence on H + concentration is difficult to predict. It has been suggested that one of the protons released on binding Fe(III) to transferrin is derived from a bound water molecule which has a p K a well below physiological p H [ 12,13]. The aquoand hydroxyferrictransferrins would not only be expected to have different conformational stabilities but also to differ in their susceptibility to attack by a chelating agent. Since a water molecule is more substitutionally labile than O H - , an increase in rate of substitution by E D T A would be expected as the p H is lowered and thus an increase in the rate of iron release. The presence of functional groups on the transferrin molecule which can undergo ionization will affect not only the apparent charge which is presented to an attacking chelate but also give species with different conformational stability as the p H is changed. N M R studies on ovotransferrin have shown that there are seven titratable histidines with p K a values from 6.42 to 7.35 which are not involved in binding G a ( I I I ) or H C O f [23,24]. Recently, Chasteen and Williams [4] have developed a model which suggests that there are two functional groups per molecule of transferrin which undergo ionization around p H 7.4 and which alter the relative iron-binding affinities of the two sites. Also, hydrogen-tritium exchange studies on transferrin [25] have indicated that conformational fluctuation is increased as the p H is lowered. Thus, the increase in the rate observed as the p H is lowered to p H 6.00 may be mainly due to the formation of conformers which are more susceptible to attack by E D T A whereas at p H values

lower than 5.6 an apotransferrin like conformer is formed and iron is released directly to the solvent. Another factor that will affect the rate of iron release is that the overall charge on the protein will become more positive at lower p H and this should aid the approach of a negatively charged ligand such as EDTA. More important however will be the charge close to the iron binding site since above p H 6.00 ionization of a functional group close to the site would greatly affect the approach of EDTA. Studies of the fluorescence energy transfer between Fe2-transferrin and various Tb(III) chelates [26] have shown that around physiological p H the sites are near electrical neutrality. No information is presently available for the individual monoferrictransferrins nor on the variation with pH. That charge is important during iron release is shown by the observation that C104 decreases the rate of release to E D T A from FeN-transferrin whereas Li + gives an increase and yet neither of these ions promotes a conformational change at the N-terminal site [6,7]. At p H 7.4 E D T A H 3 is the major E D T A species but, as the p H is lowered to 6.0, E D T A H 2- becomes predominant [5,16]. Thus decreasing the p H should favour attack of the lower charged EDTAH22although the binding strength of E D T A for Fe(III) decreases as the p H is lowered [16]. Although at low p H ( < 5.6) the relative conformational stability of the two sites appears to be the predominant factor in determining the rate of iron release, at around physiological p H it is extremely difficult to decide what role is played by each of the factors discussed above. Morgan [2] has suggested that iron release occurs after endocytotic internalization and is promoted by a lower p H within the vesicle. It is clear from the studies reported here that this mechanism would lead to a dramatic increase in the rate of iron release. However other authors [27,28] have expressed the view that endocytosis is not required for iron uptake. Although many studies [3-7] have shown clear-cut physicochemical differences between the two iron binding sites, no functional differences have been demonstrated [29,30]. This apparent paradox may be rationalized if it is assumed that the interaction of transferrin with its receptor on the membrane (see Ref. 2) leads to changes that result in functional homogeneity. Although no direct evidence

146

yet exists for such behaviour, iron release has been observed to occur homogeneously under certain conditions [5,6,31]. However, a fairly long residence time on the membrane site could also lead to apparent site homogeneity even though the two sites release iron at different rates. It is clear that much more information is required on the conformational behaviour of transferrin and on its binding reaction to the membrane site before we can gain a better understanding of the thermodynamics and kinetics of its reactions. Acknowledgements

D.A.B. would like to thank the Council for Scientific and Industrial Research, Pretoria, for financial support and Professors P. Aisen, G.W. Bates, N.D. Chasteen a n d K.N Raymond for kindly providing copies of manuscripts prior to publication. References 1 Aisen, P. and Listowsky, I. (1980) Annu. Rev. Biochem. 49, 357-393 2 Morgan, E.H. (1981) Mol. Aspects Med. 4, 1 123 3 Aisen, P., Liebman, A. and Zweier, J. (1978) J. Biol. Chem. 253, 1930-1937 4 Chasteen, N.D. and Williams, J. (1981) Biochern. J. 193, 717-727 5 Baldwin, D.A. (1980) Biochim. Biophys. Acta 623, 183-198 6 Baldwin, D.A. and de Sousa, D.M.R. (1981) Biochem. Biophys. Res. Commun. 99, 1101-1107 7 Baldwin, D.A., de Sousa, D.M.R. and Ford, G. (1981) Paper presented at the Fifth International Conference; Proteins of Iron Storage and Transport, La Jolla, CA 8 Princiotto, J.V. and Zapolski, E.J. (1975) Nature, 255, 87-88 9 Lestas, A.N. (1976) Br. J, Haematol. 32, 341-350

10 Harris, D.C. (1977) Biochim. Biophys. Acta 496, 563-565 11 Gelb, M.H. and Harris, D.C. (1980) Arch. Biochem. Biophys. 200, 93-98 12 Harris, W.R., Carrano, C.J., Pecoraro, V.L. and Raymond, K.N, (1981) J. Am. Chem. Soc. 103, 2231-2237 13 Pecoraro, V.L., Harris, W.R., Carrano, C.J. and Raymond, K.N. (1981) Biochemistry. 20, 7033-7039 14 Graham, G.A. and Bates, G,W. (1977) in Proteins of Iron Metabolism (Brown, E.B., Aisen, P., Fielding, J. and Crichton, R.R., eds.), pp. 273-280, Grune and Stratton, NY 15 Zapolski, E.J. and Princiotto, J.W. (1980) Biochemistry 19, 3599-3603 16 Gustafson, R.L. and Martell, A.E. (1963) J. Phys. Chem. 67, 576-582 17 Bates, G.W. (1981) Paper presented at the Fifth International Conference; Proteins of Iron Storage and Transport, La Jolla, CA 18 Frieden, E. and Aisen, P. (1980) Trends Biochem. Sci, 5, XI 19 Aisen, P. and Liebman, A. (1973) Biochim. Biophys. Acta 304, 797-804 20 Christensen, T.G., Comeau, R.D., Wit, D.P. and Woodworth, R.C. (1980) Ci~nc. Biol. (Portugal), 5, 113-115 21 Van Eldik, R., Palmer, D.A., Kelm, H. and Harris, G.M. (1980) Inorg. Chem. 19, 3679-3683 22 Tsuda, T., Chiyo, Y and Saegusa, T. (1980) J. Am. Chem. Soc. 102, 431-433 23 Alsaadi, B.M., Williams, R.J.P. and Woodworth, R.C. (1980) Ci~nc. Biol. (Portugal), 5, 137-139 24 Alsaadi, B.M., Williams, R.J.P. and Woodworth, R.C. (1981) J. Inorg. Biochem. 15, 1-10 25 Llinas, M. (1973) Struct. Bonding 17, 135-220 26 Yeh, S.M. and Meares, C.F. (1980) Biochemistry 19, 50575062 27 Loh, T.T., Yeung, Y.G. and Yeung, D. (1977) Biochim. Biophys. Acta 471, 118-124 28 Verhoef, N.J. and Noordeloos, P.J. (1977) Clin. Sci. Mol. Biol. 52, 87-96 29 Huebers, H., Josephson, B., Huebers, E., Csiba, E., and Finch, C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2572-2576 30 Groen, R., Hendricksen, P., Young, S.P., Liebman, A. and Aisen, P. (1982) Br. J. Haematol. 50, 43-53 31 Carrano, C.J. and Raymond, K.N. (1979) J. Am. Chem. Soc. 101, 5401-5404