An electron paramagnetic resonance study of bovine α-lactalbumin-metal ion complexes

Iron Binding by Phosvitin: Variation of Rate of Iron Release as a Function of the Degree of Saturation of Iron Binding Sites James Grogan and George Taborsky Section of Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, California

ABSTRACT The rate of iron release from Fe(III)-phosvitln complexes, at vaned degrees of saturaUon, was stu&ed Iron release was induced by reduction m the presence of the ferrous 1on chelator, ophenanthrohne. If iron release was reduced photochemically (without a chermcal reductant), the reacUons proceeded m zero order fashion, independently of the degree of saturation but with a strong dependence on the concentrauon of phenanthrohne When hydroqumone was added and the reactions were conducted m the dark, iron release followed first-order lonetlcs and the rate constants showed a clear dependence on the degree of saturation of the protein, which was most marked at lower levels of saturation The results imply control of iron release by binding site differences produced by different mtramolecular enwronments as the protein provides different combmauons of its phosphoserlne groups as hgands depending on the number of iron atoms to be accommodated per protein molecule

INTRODUCTION Phosvitin, the highly phosphorylated protein component of the yolk of vertebrate eggs, is likely to be the major metal complexing agent in the egg yolk, which is also the principal repository--discounting the shell--of the egg's metal content. There is an approximate stoichiometric equivalence between multivalent cations and proteinbound phosphorus (at 1-1.5 mmol per average yolk of the chicken egg) and both, metals and phosphoprotein, are concentrated--presumably in a structurally wellordered manner--in the organized elements of the yolk, the granules or platelets (cf. Ref. 1). Therefore, an understanding of the interactions between metal ions and phosvitin may produce insight of functional significance. Indeed, the putative metal-complexing role of phosvitin may begin even prior to its Address reprint requests to Dr George Taborsky, Section of Blochenustry and Molecular Biology, Department of Biological Sciences, Umverslty of Cahforma, Santa Barbara, CA 93106

Journal of Inorganw Btochemtstry 26,237-246 (1986) © 1986 Elsevier Science Pubhshmg Co., Inc. 52 Vanderbllt Ave , New York, NY 10017

237 0162-0134/86/$03 50

238

J Grogan and G Taborsky

deposition in the egg insofar as the precursor protein of phosv~tin, vitellogenin, is a fully phosphorylated product of the liver and its metal-complexing properties may find functionally significant expression already in the maternal organism where the protein is a circulating blood protein prior to its uptake by the oocyte. Correlations have been noted between the induction of blood phosphoprotein synthesis and blood iron levels [2]. The putatwe role of the circulating phosphoprotein as a metal carrier has been extended also to other metals such as Mg [3] and Cu [4]. The relative importance of the phosvitin precursor as a metal transporter appears to be high. One third [5], or even one half [6], of the circulating blood iron has been reported to be bound to phosphoprotein rather than transferrin m the egg-laying animal. Transferrin has been proposed to be the iron donor to vitellogenm m the course of the latter's hepatic synthesis [7]. In addition, or alternatively, ferritin has been proposed to serve as the source of phosphoprotein-bound iron, initially in the ferrous form, which would later undergo facile autoxidation m the oxygen-rich blood [8[. These observations justify concern with phosvltm as a metal complexing agent of potential biological Importance especially because relatively little is known about dynamic aspects of the phosvitin complex. In any case, it is known that as the protein is isolated from the egg, it contains 2-3 atoms of iron per molecule [9] However, its potential binding capacity is very much higher (about 60 atoms per molecule), corresponding to an Fe:P ratio of 0.5--suggesting a palrwlse involvement of phosphate groups in the binding of iron [10, 11]. (By way of contrast, it may be noted that the stoichlometry of Ca and Mg binding is 1:1 with respect to protein-bound P [12].) The binding of ~e 3+ is exceedingly strong ( g a s s o c , a t l o n = 1018 M [13]); that of Fe 2÷ is very much weaker [14]. At other than strongly acidic pH values, the Fe(II)-phosvitin complex is readily autoxidized [15]. In the autoxidized form, the iron is practically inaccessible, although it can be removed by prolonged dialysis against a strong Fe 3÷-chelator such as EDTA [16]. Our current studies address the dynamics of autoxldatively produced Fe(III)phosvitin complexes and, particularly, the circumstances of the release of iron from its strongly held state upon chemical reducnon Preliminary observations suggested that the iron binding sites of phosvitin (of the chicken as well as other species) may be nonuniform in that a propomon of the ~ron may be" more readily released than the rest, upon reduction [1]. In this paper, we present a kinetic description of the mobilization of phosvitin-bound iron and an interpretation of the data m terms of a model that would account for the suggested nonuniformity of binding sites. MATERIALS AND METHODS Chicken phosvitin was prepared according to Joubert and Cook [16] and the major component was purified by the procedure of Clark [17]. Chemicals were commercially obtained materials of analytical grade and used without purification with the following exceptions: FeSO4-7H20 (Mallinckrodt AR) crystals were selected, discarding those which showed a visible white coating indicative of some oxidative and/or hydrolytic decomposition, o-Phenanthroline (Aldrich) was recrystalhzed from an ethanol-water solution. Ferric ion "standard" solutions were prepared using analytical grade iron wire (Baker and Adamson), which was sanded, washed, dried, weighed, and dissolved in nitric acid to a final concentration of 100 mg/hter of Fe m about 0.2 N HNO3.

Iron Binding by Phosvitin

239

Fe analyses were carried out according to Sandell [18], by colorimetric determination of the Fe(II)-phenanthroline complex formed in the presence of, typically, 2.5 mM phenanthroline, 18.2 mM hydroquinone (which was omitted if preexisting Fe 2+ ion was to be determined), and less than 0.1 mM iron. The pH of the reaction mixture was about 5.5. Routinely, absorbance readings were made one hour after mixing, at 508 nm, using an LKB Ultrospec 4050 spectrophotometer and cells of 0.5 or 1.0 cm light path. "Standard curves," obtained with either ferrous sulfate or ferric nitrate solutions, were calculated by a linear least squares procedure. The average slope of nine standard curves was 11.32 _+ 0.33 absorbance units per mM Fe. The P content of phosvitin was determined as alkali-labile phosphate by an automated, continuous flow method using a Technicon system [19]. Fe(III)-phosvitin solutions were prei~ared by adding a suitable aliquot of a concentrated FeSO4 solution (up to 40 mM, freshly prepared in 10 mM HCI) to an aqueous solution of phosvitin (typically at 0.1-1 mg/mi, equivalent to about 0.3-3 mM phosvitin-bound P), the pH of which had been first adjusted to 6. This "autoxidation mixture" was stirred continuously for, typically, 30 min, which was more than sufficient for the complete oxidation and complexing of the added F e 2+ ion. The concentration of iron was expressed, as a rule, in terms of "percent saturation" on the basis of analytically determined Fe/P ratios such that 100% saturation was defined by an Fe/P ratio of 0.5. The release of Fe from its strongly bound state in the Fe(III)-phosvitin complex was brought about by the addition of an aliquot of the "autoxidation mixture" (described above) to an "iron recovery mixture," which consisted of a solution of phenanthroline and hydroquinone (typically at 2.5 and 18.2 mM final concentrations, respectively), at a pH of about 5.5. The autoxidation mixture was diluted thereby tenfold, as a rule. The development of color, due to the formation of Fe(II)-phenanthroline, was followed as a function of time by absorbance measurements at 508 nm. G~ven that the rate of formation of the colored Fe(II)-phenanthroline complex is practically instantaneous when the iron is present in the form of a simple salt (and a reductant, such as hydroquinone, is also present when the iron is in the Fe 3+ form), any delayed color development was taken as a direct reflection of some rate-limiting step in the conversion of the phosvitin-bound Fe 3+ form to the phenanthroline-bound Fe E+ form. In order to avoid likely complications produced by other ionic constituents, no buffer salts were employed: adequate pH control could be achieved by reliance on the buffering capacity of the phosphoprotein. Temperature was, typically, an ambient 21 _+ I*C. The iron recovery phase of the experiments was conducted routinely in the absence of light. When photochemical effects were investigated, the test tubes were illuminated from above with either a flood lamp (Infrared Industrial Reflector, 125 W) placed about 4O cm from the bottom of the test tubes that contained the reaction mixtures, or a uv lamp (ChromatoVue, maximal output at 254 nm) about 15 cm above the solutions. The temperature of the reaction mixtures under the flood lamp rose quickly to 37 + I°C. Calculations of reaction kinetics were made in terms of either zero-order or first-

240

J Grogan and G. Taborsky

order models. First-order rate constants were calculated on the basis of linear regression analyses (log ( a / ( a - x ) ) vs. time; a is the final concentration of recovered iron, x is the recovered iron concentration at time t), using the manufacturer's (Hewlett-Packard) program with an HP-25 calculator. Typically, analyses based on data over three or more reaction half times (ca. 90% reaction) were included in the analyses. Raw data (absorbance measurements) were corrected for "zero time" values, which represented reagent contributions and any instantly available iron. If these values would be attributed solely to iron, they would be accounted for, as a rule, by no more than about 5% of the total amount of iron added initially This small amount of apparently instantly available iron reflects, presumably, an amount which had either escaped autoxidation or was available for reduction (by the hydroqulnone) and complexation (by the phenanthroline) as readily as would be a simple iron salt.

RESULTS A N D DISCUSSION Autoxidative Formation of Fe(llI)-Phosvitin Complex and Release of Bound Iron As noted above, phosvltxn can bind up to 0.5 mol of Fe per mol of protein-bound P when presented with iron either in ferric or ferrous form. In the first case, Fe(III)phosvitin is obtained through ligand exchange with a soluble Fe(III) complex [11]; in the second case, Fe(III)-phosvltln IS the product of a facile autoxidation of Fe(II)phosvitin [10, 14]. The course of the autoxidation may be readily followed by the colorlmetric determination of any remaining ferrous Ion which forms a strong complex with o-phenanthroline. Although phosvltin, too, binds ferrous ion, this binding is weak [14] and, therefore, all iron in the reduced form can be expected to become complexed by phenanthroline. When, for example, a ferrous salt (e.g , FeSO4) is added to a mixture of phosvitin and o-phenanthroline, the iron becomes essentially all phenanthroline-bound even when the phenanthroline concentration IS barely matching the protein-bound phosphate concentration (phenanthroline/phosvitln-P = 0.9; phenanthroline/Fe = 10). The autoxidation depends on pH. At pH < 2, there is no significant reaction but the reaction rate increases with pH between pH 2 and 7 [14] At a pH of 4.4, for example, ferrous ion added to a phosvitin solution disappears at an approximately first-order rate with a half time of about 5 mm (Fig. 1, open circles). It may be noted that, given sufficient time (for example, 20 hr. Fig. 1, open squares), a small but significant fraction (about 5%) of the autoxidized iron reappears as Fe(II)-phenanthroline but only if the reaction mixture is not protected from light--as was the case in the experiment depicted in Figure 1. This photochemical reaction will be dealt with below. Much more of the phosphoproteln-bound Fe can be made to reappear as Fe(II)phenanthroline when a reducing agent is added. Following 30 min of autoxldatlon, sufficient for all of the iron to become oxidized and phosvitin-bound, the addition of hydroqulnone results in the recovery of about 40% of the iron from its protein-bound and oxidized form in one hour (Fig 1, closed circles). Complete iron recovery can be achieved over much longer periods of time (e.g., 20 hr; Fig. 1, closed triangles). Such findings--made also with a variety of other phosvitin species and coupled with the observation that the proportion of iron released in one hour decreased with increasing degrees of saturation of the iron binding sites [H--Imply that some of the phosvltinbound Iron may be held in a more readily accessible form than the rest. The

Iron Binding by Phoswtm

241

lO0

~

~ A

.

,~

B

±

50

~

-o~-___ t_

~ 0

D rn ~ I0

20 Time

3o

40

50

(re,n)

FIGURE 1. Rate of autoxidatlon of a phosvltm-FeSO4 rmxture and the recovery of Iron from its Fe(III)phosvmn complex by reduction with hydroqumone and complexmg with o-phenanthrohne. The autoxldation rate of a phoswtln-lron salt solution (containing, at pH 4 4, 0 3 mM pbosvmn-P and sufficient ~ron to produce an 89 % saturated Fe(IlI)-phosvttm complex) is depicted on the basis of the color (Asos) developed after one hour (©) and 20 hours ([Z) of interaction of ahquots of the oxldaUon rmxture with 2 5 mM phenanthrohne following the periods of autox~dat~on t~me indicated m the figure The autox~dauon tmxture was dduted about tenfold upon its addmon to the phenanthrohne solution The sohd symbols represent ~ron recoveries from Fe(HI)-phosvxtm following one hour (Q) and 20 hours ( • ) of exposure to phenanthroline and hydroqumone (18.2 mM) following autoxldatlon for the indicated times.

phosphoprotein-bound iron may be distributed among kinetically distinguishable subsets of binding sites. Mechanism of Iron Release

Although the mechanism by which a reducing agent may bring about the conversion of iron from its ferric, phosvitin-bound form to its ferrous, phenanthroline-bound form is not yet known, some of our observations limit the range of possibilities. We found that the chronological separation of the addition of hydroquinone and phenanthroline to the autoxidized Fe-phosvitin complex is without consequence. Preincubation of Fe(III)phosvitin with either hydroquinone, or with phenanthroline, for periods up to one hour before the addition of the other reagent resulted in the recovery of 35 + 5 % of the total iron present after one hour in the presence of both reagents. This is essentially the same proportion in which iron is recovered when both reagents are added at the same time. Such time gaps in the addition of hydroquinone and phenanthroline were also without consequence on the amounts of iron recovered as Fe(II)-phenanthroline over longer periods (2--48 hr). It seems clear that neither the reduction by hydroquinone nor ligand exchange with phenanthroline, by itself, is a rate-limiting component of the overall process. These findings are consistent with the possibility that the reactive entity is a quaternary complex, involving phosvitin, phenanthroline, the reductant, and iron. Given that Fe(III)-phosvitin is a very tight complex [13] and that Fe(II)phosvitin is weak and dissociates readily [14], and given furthermore that free Fe 3+ ion is "instantly" reduced by hydroquinone under the conditions of these experiments,

242

J. Grogan and G. Taborsky

TABLE

1. Photochemical Generation of Fe(II)Phenanthrohne from Fe(III)-Phosvltln m the Absence of Added Reductant a

Limb÷aem.~e'eh

None Flood lamp Ultraviolet lamp

k0 (nM man ~) . . . . . . 0 033 mM 0 197 mM 04 16 44

15 nd 56

°The protein was saturated with iron to 71%, at the proteinbound phosphate concentrationsshown, in the presence of 7 6 mM phenanthrohneat pH 6 bDetafls of illuminationare given m the Materialsand Methods section It is unlikely that the reductive generation of Fe(II)-phenanthrollne could involve either Fe(II)-phosvitln or free ferric ion per se as an mtermediate.

Photochemically Induced Release of Phosvitin Bound Iron We noted earlier (cf. Fig. 1) that even in the absence of reductant (hydroquinone), Fe(II)-phenanthrohne appears to be generated from Fe(III)-phosvitln albeit very slowly. Our search for an explanation led us to the realization that the chemical reductant may be "replaced" by a photochemical process which may involve water cleavage (and 02 generation [20] or the "activation" of organic acids added to the aqueous lron-phenanthroline solution [21]). In the presence of light, but without chemical reductant, the iron recovery from Fe(III)-phosvitm proceeds in a strictly zero-order fashion, the rate of Fe(II)-phenanthrohne appearance is linear to beyond about 95 % completion. Data in Table 1 illustrate that--depending on the light source-the effect may be more or less marked. The rate increased by two orders of magnitude, over the dark rate, when the reaction mixture was Illuminated by uv light. Table 2 compares the zero-order rate constants for the uv hght-dependent generation of Fe(II)phenanthroline from Fe(III)-phosvitin and from a simple ferric salt, Fe(NO3)3. Clearly, the presence of the protein limits the effectiveness of the photochemical process by about a factor of 2 The data also show that the reaction rate is insensitive to the concentration of Iron, at least over a threefold range The photochemtcal reaction is, howevel, sensitive to the concentration of phenanthrohne With relative phenanthroline concentrations of 1.2:4 (2.5, 5, and 10 mM), the relative reaction rates were 1:1.9.3.0 for Fe(III)-phosvltin and 1:1.8:2 3 for ferric nitrate

Release of Phosvitin-Bound Iron in the Presence of a Chemical Reductant When the reaction between Fe(III)-phosvltln and phenanthrohne IS conducted in the presence of a chemical reductant, hydroquinone, the results are markedly different whether the reaction occurs in the dark or uv light. In both cases, a new kinetic model operates: the reaction rate is first order (Fig. 2) and the effect of uv light (circles), compared with the dark reaction (triangles), IS much smaller (threefold enhancement)

243

Iron Binding by P h o s v i t i n

TABLE

2. P h o t o c h e m i c a l G e n e r a t i o n o f Fe(II)Phenanthroline from Fe(III)-Phoswtm a n d Fe(NO3)3 m the A b s e n c e o f A d d e d R e d u c t a n t as a F u n c t i o n o f the D e g r e e o f Saturation with Iron a

k0 (nM nun-4) Percent saturation b Fe(III)-phoswtm

Fe(NO3)3

60 68 74

134 119 153

17.6 35 5 53.1

aReactaon condmons were the same as given in Table 1; the protein-bound phosphate concentration was 2 mM n"Percent saturatmn" refers to the Fe(llI)-phosvmn complex For ferric mtrate, equwalent concentrations of iron were used

than in the absence of the reductant (up to a 100-fold enhancement; Table 1). The rate is, in this case, too, dependent on phenanthroline concentration (Fig. 3), but again the effect is more modest than the directly linear dependence of the strictly photochemical process. With hydroquinone present, the reaction rate merely doubled as the phenanthroline concentration was increased over a 12-fold range. Striking, however, is the dependence of the reaction rate on the degree of phosvitin saturation with iron. Figure 4 contains logarithmic representations of the iron recovery F I G U R E 2. Effect of hght on the rate of iron recovery. A tmxture of 2 mM phosvRln-P and FeSO4 sufficient for 71% saturation was allowed to autoxidlze for 30 nun The solution of the resulting Fe(III)phosvltln complex was then diluted tenfold by addition to a mixture of phenanthrohne and hydroqumone (at final concentrations of 2 5 and 18 mM, respectwely) and the rate of tron release from the phosvltln complex followed in terms of As0s measurements while the reaction rmxture was kept in the dark ( A ) or was illuminated with an ultraviolet lamp ( S ) . The corresponding first-order rate constants are 0.0106 and 0.0038 nun-1, respectively n

n

i

I

u

i

LO

o =

t

200

I

400

Time Im,n)

I

600

244

J Grogan and G. Taborsky

4 I1=

3 X

|

i

i

o

I

1 [phen I (raM)

F I G U R E 3. Effect of phenanthrohne concentration on the rate of iron recovery Conditions for the experiment were essentially those described for the dark experiment depicted in Figure 2 except that the phenanthrohne concentration was varied as shown

rates from Fe(III)-phosvltin complexes in which the degree of saturation was varied over the range of 8%-88%. At all degrees of saturation, the rates are seen to be first order but the rates are higher at low levels of saturation and they tend to approach a steady, relatively low rate as the degree of saturation approaches 100%. This is revealed more clearly in the plot of first-order rate constants vs. degree of saturation in Figure 5. The rates of iron recovery are pH-sensitive. Compared with the data shown in Figure 5--obtained at pH 5--a similar set of experiments conducted at pH 3 yielded rate constants (not shown) of nearly one order of magnitude lower, although they retained the same relationship with degree of saturation. What appeared, m our preliminary and essentially nonkinetlc observations [1] as a possibly biphasic behavior of iron recovery--a relatively extenswe iron release from F I G U R E 4. Rates of tron recovery from Fe(III)-phosvltm saturated with iron to varying degrees as shown by the numbers (% saturation) associated with the logarithmic rate plots depicted Reaction conditions were essentially those for the dark reaction m Figure 2

1.0

0

-

8

I

I

-

I

300

i

i

60(

Iron Binding by Phosvitin

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8

)

l

l

,

~

t

l

-

]

l

l

j

l

7i

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% x

5

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,

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50

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100

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Solo ration t% }

F I G U R E 5. Variation of first-order rate constants for iron recovery from Fe(IIl)-phosvitin as a function of the degree of saturatton of the phosvltan complex. Reaction conditions were essentially those for the dark reacUon m Figure 2.

sites which become filled most readily (at low percent saturation) but less extensive release from sites occupied only at high levels of saturation--may now be fitted by a different model which would also satisfy the new finding of reaction rates which vary as the initial degree of saturation is varied. The observed dependence of the rate constants on the degree of saturation implies that at low saturation a greater proportion of iron is released than at~high saturation, tending toward a constancy of the absolute velocity with which iron is released. This tendency is particularly emphasized at relatively low degrees of saturation (below about 30%--40%; Fig. 5) where the rate constants drop markedly as the initial degree of saturation is increased. At higher degrees of saturation the tendency is weakened: the rate constants vary little with the degree of saturation, meaning that the absolute velocity of iron release becomes greater as the degree of saturation approaches 1130%. Any model accounting for these observations would need to contend with the fact that as a highly saturated Fe(III)-phosvitin becomes progressively desaturated in the course of iron release, there is no switch or even gradual change in the kinetics: the rate obeys the same first-order rate constant from beginning to end for any initial ,degree of saturation. This could be explained if one assumed that the accommodation of iron by phosvitin is qualitatively different at different degrees of saturation. It is our current working hypothesis that such a variation may be related to the fact that the phosphoserine residues of phosvitin exist in a variety of intramolecular "environments," capable of providing a diversity of binding sites (and a diversity of reactive sites for the iron releasing reaction) depending on the extent to which iron must be accommodated. Relevantly to this speculation, the recently published amino acid sequence of phosvitin [22] reveals--assuming essentially all serine residues to be phosphorylated [23]--that 56% of the serines have two serine neighbors each, 36% have one, and 8 % have none. Another feature of the sequence is that 52 % of all serine

246

J. Grogan and G. Taborsky

residues which are adjacently paired are internal to given oligoserine sequences; the rest o f adjacently paired residues are at the terminals of oligoserine runs. Clearly, the potential for binding site diversity is given, if one bears in mind the 0.5 Fe/P ratio which implies pairwise cooperation of phosphate groups per site. We are now attempting to investigate the kinetics of iron release from phosvitin fragments, and simpler phosvitins from other species, which would be characterized by a lesser diversity of potential bmdlng sites. To what extent the observed behavior of iron release may be physiologically relevant is not clear. It is true that in the egg, h~gh degrees o f phosphoprotein saturation with iron are not hkely given the relatively low iron content o f the egg. However, much higher degrees of saturation may characterize the phosvltin precursor m the blood o f the maternal organism. The observed potential for " c o n t r o l " of iron release by degree o f saturation may well be relevant to this physiological condition of the phosphoprotein. Thts study was supported by research grants from the Nattonal Sctence Foundatton (PCM-80-11469) and the Umted States Publw Health Service (GM-32750). We are grateful to Mrs. Kathrm McCollum for the preparatton o f phosvmn and for phosphorus analyses.

REFERENCES 1 2 3 4

G O B J

Taborsky, Adv. Inorg. Btochem. 5, 235 (1983) Greengard, N Mendelsohn, and M Gordon, Sctence 147, 1571 (1965) Pamc, Miner. Stud Isotop Dom Atom., Panel, 1970, 81 M Reclo, J L Lattore, and J Planas, Rev Espan Ftstol. 29, 65 (1973), as m Chem. Abstr 80, 12747 (1974) 5 M A Lopez-Berjes, J M Reclo, and J Planas, PouR. Scl 60, 1951 (1981) 6 K E~ Ah atl~ W N M R~.msay, Q. J Exp~ Phystol Cog Med Sct~ 59, 159 (1974) 7 E H Morgan, Q J. Exp. Phystol Cog Med. Scl. 60, 233 (1975) 8 S Osakl, R C Sexton, E Pascual, and E Frleden, Btochem. J 151, 519 (1975) 9 D. K Mecham and H S Olcott, J. A m Chem Soc 71, 3670 (1949) 10 G Taborsky, Btochemtstry 2,266 (1963) 1l J Webb, J S Multant, P Saltman, N A Beach, and H B Gray, Btochemtstry 12, 1797 (1973) 12 K Grlzzutl and G E Perlmann, Btoehemtstry 12, 4399 (1973) 13. J Hegenauer, P Saltman, and G Nace, Btochemtstry 18, 3865 (1979) 14 G Taborsky, J Btol Chem. 255, 2976 (1980) 15 C T Grant and G Taborsky, Btochemtstry 5, 544 (1966) 16 F J Joubert and W H Cook, Can J Btochem Phystol. 36, 399 (1958) 17 R C Clark, Btoehem. J 118, 537 (1970) 18 E B Sandell, Colorlmetrtc Determmatton o f Traces o f Metals, Intersc~ence, New York, 1950, p 362 19 K McCollum and G Taborsky, Anal. Btoehem. 130, 311 (1983) 20 T C Ghkman and M E Pdhnyaeva, Ukr. Khtm Zh. 21,211 (1955) 21 J Novak, Chem Ltsty 50, 345 (1966) 22 B M Byrne, A D vatthetSchtp, I A M va~deKtundert, A C ArrLherg,M Gruher,a~dG Ab, Bsochemtstry 23, 4275 (1984) 23 R W Rosenstem and G Taborsky, Btoehemtstry 9, 658 (1970) Received October 1, 1985, accepted December 4, 1985