An Electron Paramagnetic Resonance Study of ... - ACS Publications

867

EPR Study of Vanadyl(1V)-Albumin

An Electron Paramagnetic Resonance Study of Vanadyl(1V)-Serum Albumin Complexes N. Dennis Chasteen” and Judy Francavilla Depaffment of Chemistry, University of New Hampshire, Durham, New Hampshire 03824 (Received November 12, 1975) Publication costs assisted by the Petroleum Research Fund and the National institute of General Medical Sciences

Downloaded by UNIV OF BIRMINGHAM on August 24, 2015 | http://pubs.acs.org Publication Date: April 1, 1976 | doi: 10.1021/j100549a021

This study was undertaken to examine the usefulness of the vanadyl ion (V02+)spin probe as a technique for investigating multibinding site proteins in room-temperature solutions. Quantitative EPR signal intensity measurements were made with V02+-albumin solutions at pH 5 and 25 “C where both bound and free V02+ signals could be observed. The spectrum reveals two types of binding sites. Scatchard and HughesKlotz plots reveal one “strong” binding site with a binding constant of (2.6 f 0.3) x IO6 M-l and five essentially equivalent “weak” binding sites with a binding constant of (2.5 f 0.1) X lo4 M-l. The “strong” site is probably the primary binding site for Cu2+. The binding at the “weak” sites probably occurs with carboxyl groups as suggested by the EPR parameters. The room-temperature solution spectrum of V02+ bound to albumin is very anisotropic, indicative of slow molecular tumbling. The spectrum of V02+ coordinated a t the “weak” binding sites is more rotationally averaged than a t the “strong” site. This suggests that there is some local motion at the weakly coordinating sites with a correlation time short compared to that of the protein as a whole. The spectra of V02+ doped powder albumin samples were also examined.

Introduction Of all the oxidation states of vanadium, V(1V) as the vanadyl ion (V02+)is the easiest to study in biological systems because of its characteristic EPR spectrum. Recently vanadyl ion EPR has been successfully used to probe liquid crystals,l the hydrolysis of ATP? anionic surfaces of acidic ,~ and the lipid bilayers from H. C u t i r ~ b r u m micelles: metal sites of a number of protein^.^-^ Early work suggests that this ion might also serve as a probe of nucleic acids.1° In their recent study of vanadium toxicity and molybdenum utilization in rats, Rajagopalan and co-worker& found vanadium as protein bound V02+, concentrated to about 10 ppm in the liver. In light of the growing interest in the use of the vanadyl ion as a probe of biological systems, we have undertaken a room-temperature solution investigation of vanadyl serum albumin complexes. This multibinding site protein has often served as a model for NMR and EPR spin probe and metal ion i n v e s t i g a t i ~ n s . l ~Here - ~ ~ we report for serum albumin the number of vanadyl ion binding sites, their respective stability constants, and the relative motional freedom at these sites. The tentative identity of the coordinating ligands a t these sites is inferred from the EPR parameters. The limitations of using EPR spectroscopy to obtain stability constant data are explored. Albumin is a common protein in vertabrates, but where most common proteins are specific to one function, albumin has several.12 In addition to transporting fatty acids and bilirubin, albumin apparently serves as a scavenger of heavy metals.12 In the past three decades, a vast amount of research into the binding of various moieties by albumin has been conducted. The list of metal ions used is extensive; studies have been conducted on bovine or human serum albumin using Cu2+,13-15Zn2+,16 Mn2+,17 Co2+,14 Ni2+,14J5 Cd2+,16c Gd3+,1*T13+,19 and Hg2+,19 among others. The binding properties are metal ion dependent. A vanadium study has never been reported. Experimental Section Bovine serum albumin, crystallized and lyophylized, was obtained from Sigma Chemical Co. (catalogue No. A4378).

To remove paramagnetic impurities, tentatively identified as copper and iron, M solutions of the protein were dialyzed at 4 “C against 0.01 M o-phenanthroline, pH 6, for 12-16 h followed by several changes of distilled deionized water for 2 to 3 days or until all the orange color was removed. The albumin concentration was determined spectrophotometrically, e 4.6 X lo4 cm-l M-l at 279 nm.20 Procedures for handling the vanadyl ion and avoiding contaminating metal ions were as described previ~usly.~ A 0.01 M lutidine-”03 buffer, pH 5.01, was employed in the V02+-albumin binding studies. This buffer does not coordinate to the free vanadyl ion, thus avoiding corrections for buffer coordination in the stability constant determinations. Polycrystalline samples were prepared by soaking 100 mg of BSA in 1ml of 3 M ammonium sulfate and 5% EDTA solution for 24 h followed by washing with three 1-ml portions of 3 M (NH4)2S04. Then 1ml of 3 M (NH&S04, pH 5.1 adjusted with NaOH, was added and the serum stoppered test tube purged with nitrogen before addition of an aliquot of vanadyl stock solution to give V02+: BSA mole ratios ranging from 1:l to 20:l. After soaking the solid albumin for 24 h at 4 O C , the supernatant solution was removed and the pH measured. The solid was washed a minimum of three times with oxygen-free ammonium sulfate to remove any unbound V02+ ion. The solid was then dried with a stream of nitrogen gas. In another procedure, solid samples were prepared in a similar fashion except 40% ethanol in water was used in place of 3 M (NH4)2S04. Powder samples were analyzed for vanadium by EPR spectroscopy. After dissolving the wet sample in 1 ml of distilled deionized water, the protein concentration was determined spectrophotometrically. Then 50 ~1 of concentrated HC1 and a few milligrams of ascorbic acid were added. The vanadium was determined by measuring the EPR signal height of the acid released VO(H20)52+as detailed elsewhere.6 Most EPR spectra were recorded on a Varian E-9 spectrometer operating at X-band frequency (9.5 gHz). Additional details of the experimental procedures can be found in ref 21. The Journal of Physical Chemistry, Voi. SO,No. 8, 1976

N. D. Chasteen and J. Francavilla

n

3100 G

A

SPECTRUM A "-,

L

D

c

s

5

.

0

5

5

PH


Figure 1. Spectrum A. First derivative EPR spectrum of 5.0 X M serum albumin in 0.01 M lutidine-nitric acid buffer, pH 5.0, with 9.0 equiv of VOS04 added, 25 OC. The arrows indicate contributions from VO(H20)52+. Spectrum B. First derivative EPR spectrum of VO(H20)52+at 4.42 X M concentration, pH 4.87 adjusted with NaOH, 25 OC. _

_

I

,

I '

"

Figure 3. EPR first derivative signal height divided by the instrument gain setting for 1:l V02+-albumin, 7.6 X M, as a function pH. Data for the MI = -7/2 and -512 parallel lines of protein bound V02+ are plotted.

I

EQIJIVALENTS

Figure 2. First derivative EPR spectrum of 4.9 X M BSA with 10.0 equiv of VOS04 added, pH 5.8 adjusted with NaOH. The arrows denote the labeling of the four low-field "parallel" lines. The MI = -1/2* line is actually a composite of parallel and perpendicular components. At this pH, the unbound VOz+ exists as a hydroxide which does not exhibit a room-temperature EPR spectrum.

Results and Discussion Binding Constants. The room temperature spectrum of a pH 5 solution containing V02+ albumin in a molar ratio of 9:l is shown in Figure 1A. The spectrum consists of a superposition of resonances from free and bound V02+. The eight arrows in Figure 1A denote the eight resonances of unbound VO(Hz0)b2+ (Figure 1B). The remaining resonances constitute the spectrum of the protein bound vanadyl ion. Increasing the pH to 5.8 results in a loss of the VO(H20)b2+ lines due to formation of a VO(OH):! precipitate which exhibits no EPR spectrum at room temperature.22 A room temperature solution spectrum of albumin bound V02+ a t pH 5.8 is shown in Figure 2. The spectrum displays parallel and perpendicular features characteristic of frozen solution or polycrystalline samples; upon binding the motion of the vanadyl ion becomes highly restricted and is largely governed by the slow tumbling of the albumin molecule. In Figure 3 the first derivative intensities of the two lowThe Journal of Physical Chemistry, Vol. 80, No. 8, 1976

Leo2' ADDEC

Figure 4. EPR first derivative signal height of protein bound V02+ as a function of equivalents of VOS04 added. 5.0 X M serum albumin, 0.01 M lutidine-nitric acid buffer, pH 5.0. Data for the MI = -5.2 and -1/2 "parallel" lines are plotted.

field "parallel" lines M I = -5/2 and -7/223 (Figure 2) are shown as a function of pH for a 1:l VOZf-albumin complex. The first derivative intensities are proportional to the amount of V02+ ion bound to the protein if no line width variation occurs over the pH range. This is the case here. Binding is seen to maximize and level off at about pH 4.7. Subsequent experiments were done at pH 5.0 which enabled us to measure the free V02+ ion concentration. At a higher pH the VO(H20)52+spectrum could not be observed because of metal hydroxide formation as noted above. This is a limitation of the method. By measuring the intensity of the EPR lines of the VO(H20)52+ and V02+-albumin species as a function of the V02+/albumin mole ratio, one can determine the number of binding sites and their respective binding constants. Figure 4 displays the signal intensities of the M I = -512 and -1/2 parallel lines (see Figure 2) as a function of the number of equivalents of V02+ added to the protein at pH 5.01. Similar plots (not shown) are obtained with the other lines in the spectrum. The plots show a first break at an average value of 1.2 equiv and a second break a t about 5.5

869


2

4-

8

6

EQUIVALENTS V02+ ADDED


Figure 5. Concentration of VO(HZO)~~+ as a function of equivalents of VOSO4 added to the albumin solution described in Figure 4.

5‘ Figure 7. Scatchard plot after substracting from the data the effect of the binding at the first site. The deviation from the line at high V‘ values is due to vanadyl hydroxide formation. See text for definition of quantities.

I

2

I

I

I

3

L

5

3

Flgure 6. Scatchard plot showing at least two classes of binding sites for the titration of albumin with VOZf. See text for definition of quantities.

equiv; this indicates that chelation takes place at one “strong” binding site followed by four to five “weaker” binding sites. The abrupt change in slope a t 1.2 equiv is a consequence of the different relaxation behavior of the V02+ ion a t the two types of sites. There appears to be greater motional freedom at the “weak” sites; this will be discussed in more detail later. The concentration of free VO(H20)52+, measured from its EPR first derivative intensity,24 as a function of equivalents of V02+ added is shown in Figure 5. The curve begins to roll over at high equivalent values due to the appearance of a VO(OH)2 precipitate in the titration vesseLZ4 In the absence of hydroxide precipitation, the curve in Figure 5 would climb steeply at high equivalent values. In the binding of V02+ ion to albumin it is apparent that multiple equilibria are involved. Data were treated by the method of Scatchard et al.25Plots of ~rl[VO~+]f,,,vs. 5 were

made where ~ris the moles of V02+ bound per mole of protein and [V02+]fr,, is the VO(H20)b2+concentration. Ideally, one would like to measure the concentration of the vanadyl protein complex ,from its spectrum shown in Figure 2. Unfortunately, one does not know the proportionality constant relating the peak-to-peak signal intensity of the first-derivative curve to the concentration. Nor is it practical to perform a double integration because of the myriad of fine structure in the spectrum. Some of the problems of qualitative analysis by EPR spectroscopy are discussed in ref 6. Instead, the concentration of V02+ bound to the protein, [VO2+]bound, was determined from the relationship [VO2+]bound = [Vo2+],dded - [V02+]f,,,. This equation is valid only in the absence of vanadyl hydroxide formation, i.e., the early part of the titration of metal free albumin with Vo2+. 5 is given by Ir = [V02+]b,und/[albumin]. A Scatchard plot of the data (Figure 6) shows two straight line regions which indicates a t least two classes of binding sites.25The intercept of the dashed curve with the abscissa yields a value of n l = 1.25 for the number of binding sites in the first class; correspondingly the slope, -K1, gives the binding constant K1 = 2.3 X lo6 M-l. Other titration data not presented here confirm that there is only one binding site in the first class.21Hereafter we refer to this as the “strong” site, although the interaction is a relatively weak one. The leveling of the curve to zero slope a t high ~rvalues in Figure 6 is a consequence of the competition of OH- with the protein for the available V02*. Data points-are reliable only up to 5 N 3. To determine the number of sites in the second class and their binding constant, we plot Y ’ / [ V O ~ +vs. ] ~U’~ where ~ ~ 5‘ = ij - 1 (Figure 7 ) . This procedure substracts out the contribution of the “strong” site to the data. This is valid only when the two classes of sites are noninteracting, i.e., binding at one site does not affect binding a t another. This seems to be the case here. From Figure 7 we obtain n2 = 5.0 and K2 = 2.5 X lo4 M-l for the second class of sites, the “weak” sites. Again, the deviation of points above 3’ = 2 is a consequence of the onset of hydroxide precipitation. The Journal of Physical Chemistry, Vol. 80,No. 8, 1976

870

N. D. Chasteen and J. Francavilla

TABLE I: EPR Parameters of Frozen Solutionsa

Site

Ao( i1.O) b*c

A 11 (A0.5)“

A 1(f1.0)‘

go(f0.002)d


Strong 100.3 172.8 64.0 1.966 Weak 102.8 177.1 65.6 1.965 a 0.01 M lutidine-”02 buffer solution, DH5.01. Calculated from A0 = (All + 2A1)/3.

g 11 (f0.001)

gl(f0.002)

1.939 1.938 Units of lo-* cm-l.

1.979 1.979 Calculated from

ments and imidazole is known to coordinate to V02+ in These data were also plotted according to the method of several other protein^.^,^^^ Hughes and Klotz26which yielded n l = 1.3 with K1 = 2.8 X In addition, the vanadyl ion coordinates very weakly to lo6 M-l and n2 = 5.0 with K2 = 2.5 X lo4 M-l. These a-amino groups. A formation constant of only 1.8 is obvalues are in good agreement with the results from the served for the formation of a bidentate 1:l vanadyl-glycine Scatchard plots. complex from the ring closure of the monodentate oxygen Nature of the Binding Sites. The primary site for copper coordinated complex.27 The low affinity for amino groups binding is located on the amino terminal end of the prois further amplified by the lack of reports in the literature tein12J3 i.e., Asp-Thr-His-Lys. In this strong site, the aof vanadyl complexes with aliphatic amines. The a-NH2 amino nitrogen, the first two peptide nitrogens, and a nigroup in serum albumin has a pK, = 7.8.28Finally, ionizatrogen of the imidazole group of His-3 coordinate in a square-planar arrangement about the c ~ p p e r ~ ~ Ni2+ * , ~ J ~ tion and coordination of peptide amide linkages is usually found only for c0pper.~9 competes effectively with Cu2+ for the same binding site, Molecular Motion. Except for small differences in line apparently because Ni2+ readily forms square-planar comwidths and hyperfine splittings, room-temperature EPR plexes.14J5 spectra of solutions of vanadyl labeled proteins are similar We have conducted similar experiments with V02+-alto spectra obtained with frozen solution or polycrystalline bumin and Ni2+. (Competitive binding studies with V02+ samples. For example, the values of All and A1 are 195.7 and Cu2+ were not undertaken because of the tendency of and 71.0 G, respectively, from frozen solution spectra of the these ions to undergo a redox reaction with one another.) weak sites of albumin compared to 189.3 and 74.5 G a t Varying amounts of Ni2+ were added to solutions of 4.4 X room temperature. These differences are due to partial roM 1:l V02+:albumin, 0.02 M lutidine, pH 5.0. After tational averaging of the anisotropic dipolar contribution incubation for 0.5 h the EPR spectrum was recorded. The to the hyperfine tensor in room-temperature solutions. relative intensity of the free V02+ spectrum increased from The dipolar contribution, A,, to the hyperfine splitting is 1.0 to 1.8, 2.0, and 2.5 for 1, 2, and 3 equiv of Ni2+ added, given by A , = (All - Ao)/2 where A0 = (All 2AL)/3. respectively. Thus Ni2+ and V02+ compete effectively for We define an “order parameter” S as the same strong site which is probably the copper binding site. S = A,’A o/A ,Ao’ The EPR parameters for the “strong” and “weak” sites where the primed and unprimed symbols refer to the roomas obtained from computer simulation5 of frozen solution temperature and frozen solution values, respectively. The spectra are given in Table I. The parameters for the above definition is equivalent to that of Hubbell and “weak” sites are very similar to those for V02+ bound extraneously to single carboxyl groups in polycrystalline samMcConnell for describing rapid anisotropic motion in ples of bovine insulin5 and carboxypeptidase A.8 The m e r n b r a n e ~ Here . ~ ~ we use S as a starting point for discussion of motion in vanadyl proteins where the metal ion “weak” sites are probably monodentate coordinating cartumbling is primarily governed by the isotropic rotational boxyl groups of glutamyl or aspartyl amino acid residues. If one assumes that Cu2+ and V02+ bind at the same , the protein as a whole. For rapid correlation time, T ~ of ~ s) A,’ is essentially averaged to zero tumbling ( T < strong site, the question arises as to whether all four nitrogen ligands (a-amino, imidazole, and two amide) also coorand S is zero. Conversely in a completely “immobilized” dinate to the vanadyl ion. The evidence is most consistent spectrum ( T > ~ s) A,’ equals A, and S is one. The facwith only the imidazole group of His-3 binding in the first tor Ao/Ao’ is used to roughly correct for variations in spin density from solvent effects due freezing the ~ample.~O For coordination sphere. The hyperfine splittings of the “strong” site are not greatly different from those of the the vanadyl ion, this correction is generally near unity. One expects S to vary monotonically with the correlation “weak” site although the former values are smaller as extime for isotropic motion. The correlation time can be estipected for nitrogen vs. oxygen coordination (Table I). The mated from the Debye r e l a t i ~ n s h i pT~ , ~= ~ VqkT, in which similarities in the EPR parameters probably reflect the fact that most of the ligands for both sites are water molethe protein is treated as a sphere of volume V rotating in a cules. Application of the rule of average environment for fluid medium (H2O) with a viscosity q at T = 298 K. 12 is three water molecules and one aromatic nitrogen donor the Boltzmann constant. We calculate an approximate molecular volume from the partial specific volume of 0.74 coordinated equatorially5 leads to a predicted isotropic hyml/g. Under these assumptions, the rotational correlation cm-I which is not perfine splitting A0 = 101.8 X time is given by T~ = 3.0 X W s, where W is the mogreatly different from the experimental value of A0 = 100.3 lecular weight (g/mol) of the protein. X 10-4 cm-l (Table I). Inclusion of other nitrogen donors Table I1 lists values of S and estimated T~ for some vanain the first coordination sphere leads to lower A0 values dyl-protein complexes of different molecular weight. Aland considerably poorer agreement. Clearly, this procedure though the values of S have an experimental uncertainty of is not sufficiently reliable by itself to draw firm concluabout f O . O 1 , they do show a definite increase with increassions. However, the pK, N 6 for imidazole is the closest of ing molecular weight and estimated T ~ .I t is clear, however, the nitrogen donors to the pH 5.0 used in these experi-

+

The Journal of Physical Chemistry, Vol. 80, No. 8, 1976

87 1


TABLE 11: Order Parameters (5')for Vanadyl-Protein Complexes Protein

Molecular wt, g/mol

Tr,10-8S

Serum albumina 68 000 (weak sites) Carbonic 31 000 0'94 anhydrase Carboxypepti35 000 1.1 dase AC Bovine serum 68 000 2.1 albumina (strong site) Transferrind 80 000 2.4 a This work. Reference 7 . Reference 8. J. C. and N. D. Chasteen, to be submitted for publication.


that

s 0.919 0*930 o.936

fate procedure revealed additional resonance lines indicative of V02+ binding in at least three different chemical environments. At these high molar ratios binding at various weakly coordinating sites in the solid state begins to occur.21

Acknowledgment. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the National Institute of General Medical Sciences, Grant No. GM20194-03, for support of this research.

0.952 References and Notes 0.956 Cannon

s values (or other quantities such as A ll'/A 1132) based on

hyperfine Of room-temperature vanadyl protein solutions, are not sufficiently sensitive in the tirne domain T1. 5 10-8 to permit dne to obtain accurate correlation times. For nitroxide spin labels, in which the hyperfine anisotropy is an order of magnitude smaller than for vanadyl, this limitation sets in a t a larger correlation time, T~ 2 10-7 s.32 The order parameterfor the weak sites in vanadyl-albumin is significantly smaller than for the strong site, 0.919 vs. 0.952 (Table II), which suggests localized motion a t the weak sites which is fast relative t o the tumbling rate of the protein as a whole. This is reasonable in view of the small binding constant which probably reflects monodentate coordination to a single carboxyl group a t these sites. The EPR parameters discussed earlier (Table I) are also consis'tent with this.5 Powdered Samples. Powdered samples of albumin were soaked in ammonium sulfate solution, p~ 5 , containing sufficient vanadyl sulfate to give a W d i u m to protein molar ratios from 1:l to 20:l in the soaking solution (see ~ ~ section). powder ~ samples ~ removed from. ~ soaking solutions of molar ratios 1:l to 5:l exhibited spectra indicative of only one typeof binding site with paramecm-l, A i = 63.1 X ters Ail = 170.8 X cm-', gil = 1.936 and g l = 1.978. These parameters are similar to those of the strong site in frozen solutions of the protein (Table I). Metal analysis of a sample from 2:1 soaking solution gave 1.04 mol of vanadium per mole of albumin. Thus, powder and frozen solution samples appear to exhibit similar binding properties involving one "strong" site. In contrast, the primary site in powder Obtained from the ethanol-H2O soaking procedure has quite different parameters ( A l = , 166.3 x 10-4 cm-l, A l = 62.2 x 10-4 ern-', glt = 1.942 and g l = 1.978) from those of fmZen d u tion samples or powder samples from the ammonium sulfate the less polar effects the surface of the protein and induces a quite different mode of binding of the vanadium. Spectra of powder samples obtained from the rather high molar soaking ratios of 10:l and 2O:l of the ammonium SUI-

(1) J. P. Fackler, J. D. Levy, and J. A. Smith, J. Am. Chem. SOC.,94,2436

(1972). (2)G. M. Wolterman, R. A. Scott, and G. P. Haight, J. Am. Chem. SOC.,96, 7569 (1974). (3) W. 2.Plachy, J. K. Lanyi, and M. Kates, Blochemistry, 13,4906 (1974). (4)P. Stilbs and B. Lindman, J. Colloidlnterface Scl., 46, 177 (1974). (5) N. D. Chasteen, R. J. DeKoch, B. L. Rogers, and M. W. Hanna, J. Am. Chem. SOC.,95, 1301 (1973). (6)J. J. Fitzgerald and N. D. Chasteen, Anal. Biochem., 60,170 (1974). (7)J. J. Fitzgeraid and N. D. Chasteen, Biochemistry, 13,4338 (1974). (8) R. J. DeKoch, D. J. West, J. C. Cannon, and N. D. Chasteen, Biochemlstry, 13,4347 (1974),Corrections, ibid., 14,196 (1974). (9)J. c. Cannon and N. D.Chasteen, Biochemistry, 14,4573(1975). (10)W. Snipes and W. Gordy, J. Chem. Phys., 41,3661 (1964). (11)J. L. Johnson, H. J. Cohen, and K. V. Rajagopaian, Biochem. Biophys. Res. Commun., 56,940 (1974). (12)T. Peters, Adv. Clln. Chem., 13,37 (1970). (13) (a) W. T. Shearer, R. A. Bradshaw, F. R. N. Gurd, and T. Peters, J. Biol. Chem., 242, 5451 (1968);(b) F. H. Reynolds, R. K. Burhard, and D. D. Mueller, Biochemistry, 12, 359 (1973);(c) J. C. Cassatt, J. Biol. Chem., 248, 6129 (1973);(d) B. Sarkar and Y. Wigfield, Can. J. Blochem., 46, 601 (1968);(e) S.J. Lau, T. P. A. Kruck, and 9.Sarkar, J. Biol. Chem., 249,5878 (1974),and references therein. (14)I. M. Kolthoff and B. R. Willeford, J. Am. Chem. Soc., 80, 5673 (1958). (15) T. Peters and F. A. Blumenstock, J. BiOl. Chem., 242, 1574 (1967). (16)(a) R. Osterberg, Acta Chem. Scand., 25, 3827 (1971);(b) E. L. Giroux, Biochem. Biophys. Acta, 273, 64 (1972);(c) M. S. N. Rao and H. Lal, J. Am. Chem. SOC.,80,3222(1958). (17)A. S.Mildvan and M. Cohn, Biochemistry, 2,910 (1963). (18)J. Reuben, Biochemistry, IO, 2834 (1971). (19)J. L. Sudmeier and J. J. Pesek. Anal. Biochem., 41,39 (1971). (20)H. A. Soper and R. A. Harte, Handbook of Biochemistry", Chemical Rubber Company Press, Cleveland, Ohio, 1973,p C71. (21)J. Francavilla, M.S. Thesis, University of New Hampshire, Durham, N.H.,

1974. (22)J. Francavilla and N. D. Chasteen, lnorg. Chem., 14,2860 (1975). i assignment assumes ~ ~ ~ ~ (23)This a negative vanadium nuclear hyperfine coupling constant. Nuclear spin, i = 712. (24)For a discussion of the validity of this procedure, see ref 6. in the early M VOS04 stock solution with a stages of the titration, addition of a microliter syringe produced a high localized concentration of vanadyl ion resulting in a VO(OH)* precipitate which dissolved upon gentle agitation. As the titration progressed, the precipitate dissolved more slowly until at 3 equiv of V02+ added, it would not completely dissolve with limited stirring. Extensive agitation of the solution to affect complete dissolution was not possible because of protein denaturation from froathing. In principle, all the precipitate should dissolve since the VO(IV) ion is soluble to 1 X M at pH 5: for VO(OH)2, Ksp = 1.08 X The highest free V02+ ion concentration achieved in these experiments was only 6 X M. (25)G. Scatchard, J. S. Coleman, and A. L. Shen, J. Am, Chem. SOC.,79,

12(1957). (26)T. R. Hughes and I. M. Klotz, Methods Biochem. Anal., 3,265 (1956). (27)H. Tomiyasu and G. Gordon, J. Coord. Chem., 3,47 (1973);, (28)K. Linderstrom-Lang and S. 0. Nlelson, "Acid-Base Equilibria of Proteins" in "Electrophoresis: Theory, Methods, and Applications", M. Bier, Ed.. Academic Press, London, 1959,p 49. (29)J, Peisach, p, Aisen, and W, E, Blumberg, Ed,, "The Biochemistry of Copper", Academic Press, New York, N.Y., 1966,pp 19 and 87. (30)W. L. Hubbeli and H. M. McConneil, J. Am. Chem. SOC.,93,314 (1971). (31)N. Bioembergen, E. M. Purceil, and R. V. Pound, Phys. Rev., 73, 679 ( 1948). (32)R. P. Mason and J. H. Freed, J. Phys. Chem., 78,1321 (1974).

The Journal of Physical Chemistry, Vol. 80, No. 8, 1976

An Electron Paramagnetic Resonance Study of ... - ACS Publications

Recommend Documents