Eur. J. Biochem. 225, 363-368 (1994) 0 FEBS 1994

A new structural transition of serum albumin dependent on the state of Cys34 Detection by 'H-NMR spectroscopy

John CHRISTODOULOU, Peter J. SADLER and Alan TUCKER Christopher Ingold Laboratories, University of London, England

(Received May 16/July 8, 1994) - EJB 94 0702/3

1. Reactions of fatty-acid-free bovine serum albumin and recombinant human albumin with a range of antiarthritic gold(1) complexes [auranofin, deacetylated auranofin, triethylphosphinegold(1) chloride] and related thiols (thioglucose, tetraacetylthioglucose, glutathione, dithiothreitol) have been investigated using 'H-NMR spectroscopy.

2. In reactions of albumin with auranofin, tetraacetylthioglucose and dithothreitol, release of cystine was detected, whereas for deacetylated auranofin, thioglucose and glutathione, mixed disul- phides with cysteine were produced. It has been previously proposed that Cys34 of human and bovine serum albumins is partly blocked by disulphide formation with cysteine and glutathione. The above reactions lead to deblocking by thiol-disulphide interchange reactions. No release of glutathione from albumin was detected.

3. Changes in the His HE^ regions of the 'H-NMR spectra show that albumin exists in two structural forms dependent on whether the side-chain of Cys34 is a free thiolate, or blocked by gold(I)triethylphosphine, by disulphide formation with cysteine or by another form of oxidation. We propose that Cys34 is either in a buried or in an exposed environment; the possible molecular basis of the structural change is discussed.

4. The relationship between reactions at Cys34, cysteine release, and the observed structural transition are discussed in terms of chrysotherapy, albumin metabolism and the use of gold(1) as a heavy atom derivative in X-ray crystallographic studies of albumins.

Serum albumin is the major plasma protein, accounting for about 60% of the total protein in blood serum, with a concentration of about 42 g 1-' (0.63 mM) [I]. Albumin is a single-chain protein of about 580 amino acids (66.5 kDa) containing 35 cysteine residues formed into 17 structural di- sulphide bonds plus one free thiolate (Cys34). The protein is composed of three structurally similar domains [2, 31, each

Correspondence to P. J. Sadler, Department of Chemistry, Birk- beck College, University of London, Gordon House, 29 Gordon Square, London, England WClH OPP

Fax: +44 71 380 7464. Abbreviations. Alb-'Cys'34-S-, albumin with Cys34 in the free

thiolate form ('Cys'34-S is used to allow reference to modifications of the sulphur of Cys34 of albumin without implying addition of further sulphur) ; Alb-'Cys'34-SCys, albumin with Cys34 blocked by disulphide formation with Cys ; Alb- 'Cys'34-SAuPEt3, albumin with triethylphosphine gold(1) bound to Cys34; Alb-'Cys'34-SOXH, albumin with Cys34 in an oxidised form; BSA bovine serum albu- min; COSY, homonuclear shift-correlated spectroscopy ; m y s , cystine; m G l c , disulphide formed from cysteine and thioglu- cose; ID and 2D, one-dimensional and two-dimensional; Et,. PAuSGlc, triethylphosphine gold(1) thioglucose; Et,PAuSGlcAc,, triethylphosphine old(1) (2,3,4,6-tetra-acetyl)-l-~-~-thioglucose

one and cysteine ; Glu(-Cys-Gly), glutathione (y-glutamylcysteinyl- glycine); HSA, human serum albumin; HSGlcAc,, (2,3,4,6-tetra- acetyl)-l-~-D-thiog~ucose; pH*, pH meter reading in D,O solution ; NaSGlc, sodium 1 -P-D-thioglucose; rHA, recombinant human albu- min; TOCSY, total shift-correlated spectroscopy.

(auranofin) ; Glu(- F--T ys-Gly) ys, disulphide formed from glutathi-

of which contains two sub-domains. Isolated samples of the protein typically have less than 1 mol SWmol albumin, con- sistent with the blocking of Cys34 of some protein molecules via disulphide formation with endogenous thiols, usually cited as cysteine and glutathione [l]. Cys34 is thought to have an unusually low pK, value, probably between 5-7 [4], and is located in a crevice in the protein structure [3].

Albumin plays a central role in the molecular pharmacol- ogy of gold drugs used in the treatment of rheumatoid arthri- tis, carrying about 80% of the circulating gold in blood [5] . Two types of gold drugs are currently used: Au(1) thiolates such as aurothiomalate given by injection, and the orally ad- ministered Au(1) triethylphosphine complex auranofin. It is possible that gold binding influences the metabolism of albu- min in rheumatoid patients and that this forms part of the therapeutic process. Gold(1) is known to have a very high affinity for thiolate sulphur ligands and the primary binding site on albumin is thought to be Cys34 [6].

We have previously used one- and two-dimensional 'H- NMR spectroscopy to investigate the chemistry of serum al- bumin in solution. Resonances have been assigned for 80 spin systems to amino acid types in bovine serum albumin (BSA) and sequence-specific assignments made for the three N-terminal amino acids of human, bovine, porcine and rat serum albumins [7, 81. We have shown that the binding of Cu2+, Ni" and Co2+ to the N-terminal binding site of human serum albumin (HSA) and BSA can be studied [9] and have

364

characterised the pH-induced structural transitions of BSA [lo].

We have recently shown that ‘H-NMR spectroscopy can detect changes in the protein structure on addition of the gold drug auranofin (Et,PAuSGlcAc,, I) or its metabolite triethyl- phosphine gold(1) thioglucose (Et,PAuSGlc, 11) to BSA along

CH,OAc

AcO

PEt, Au ‘ Aco-X5&S‘ OAc

(1)

CH,OH

HO OH Au

‘PEt,

H o T g L S ,

with the release of cystine (with auranofin) or a mixed disul- phide CySGlc (with the metabolite) [ll]. We now report a more detailed characterisation of this structural transition through studies of the interaction of a range of gold com- plexes, and related thiolate ligands, with bovine and human albumins.

EXPERIMENTAL PROCEDURES Materials

Bovine serum albumin was purchased from Boehringer (775835). Recombinant human albumin (rHA, from yeast) was the gift of Delta Biotechnology Ltd (Nottingham, Eng- land). This protein has been shown to be structurally iden- tical to serum-derived HSA by X-ray crystallography [3]. Deuterated phosphate solutions were prepared by freeze- drying 2H,O solutions containing NaH,PO, . 2H,O and Na,HPO, . ’H,O (BDH AnalaR). C‘H,O’H was obtained from Aldrich.

Et,PAuCl, sodium l-b-D-thioglucose (NaSGlc), (2,3,4,6- tetra-acety1)-1 -P-D-thioghcose (HSGlcAc,), dithiothreitol and glutathione were obtained from Sigma; Et,PAuSGlc was prepared in situ by mixing Et3PAuC1 and NaSGlc at a 1 : 1 molar ratio (in the same deuterated phosphate solution used for albumin sample preparation). Auranofin was the gift of Smith Kline Beecham (Philadelphia).

NMR spectra

Spectra were recorded at 310 K on either a Bruker AM500 or Varian Unity VXR600 spectrometer (MRC Bio- medical NMR Centre, Mill Hill) as previously described [8, 91.

Typically samples consisted of 2 mM albumin in 10 mM deuterated phosphate solution, which were then freeze-dried and redissolved in ’H,O. The pH meter reading in ’H,O @H*) of the sample was determined before, and always after NMR measurements. A Corning 145 pH meter with an Ingold pH electrode (6030-02) was used. Chemicals shifts are refer-

enced to 3-(trimethyl~ilyl)-(2,2,3,3-~H,)propionate, via in- ternal dioxan (3.764 pprn).

Gold complexes and thiols were added as microlitre ali- quots of freshly prepared concentrated solutions ( ~ 2 5 mM) in 10 mM deuterated phosphate solution or in C2H,02H for Et,PAuSGlcAc,, Et,PAuCl and HSGlcAc,. Control additions of C2H302H to BSA and rHA showed no significant changes in the spectra at concentrations of C2H,0’H up to 10% by volume. Spectra were normally recorded within an hour after additions were made, or 24 h if two-dimensional (2D) data were obtained. The thiolate contents of BSA and rHA were estimated using Ellman’s reagent, 5,5’-dithiobis(2-nitroben- zoic acid) 112).

RESULTS

We have used ’H-NMR spectroscopy to study reactions of gold(1) complexes and thiols with BSA and rHA at various molar ratios, at pH* 7 or pH* 6.4 in 10 mM sodium phos- phate at 310 K. No attempt was made to study kinetics but, in general, reactions appeared to reach equilibrium within the time taken to prepare for the NMR measurements (15- 30 min). Formation of gold-albumin complexes was con- firmed by 31P NMR and by the appearance of cross-peaks in 2D ‘H homonuclear shift-correlated (COSY) spectra or total shift-correlated (TOCSY) spectra at 1.19- 1.95 ppm assign- able to Et,PAu’ bound to albumin at Cys34 (data not shown).

The interpretation and assignment of one-dimensional (1D) spectra was greatly aided by the use of resolution en- hancement (exponential and sine-bell functions [8], as in Figs 1-3) and by acquisition of 2D COSY spectra for se- lected samples. The use of resolution enhancement usually makes comparison of intensities unreliable in spectra con- taining resonances of widely differing line-widths. However, in this work, all peak height measurements are of two peaks (n and n: see below) which appear to have similar line- widths.

Changes in several regions of the spectra accompany these reactions. In the His HE^ region (7.5-8.5 ppm) several resonances are affected but the most significant changes are for resonances assigned to His3. We have previously shown in ‘H-NMR spectra of BSA [lo] that His3 exhibits two reso- nances (n and n? above pH* 6 which are in slow exchange on the NMR timescale. We have now confirmed (unpub- lished results) that two resonances are similarly observed above pH* 6 for His3 in rHA. A new broad resonance is seen at -0.35 ppm when gold is bound and is probably as- signable to a methyl group which is near to the face of an aromatic ring.

Gold complexes

On addition of Et,PAuSGlcAc, or Et,PAuCI to solutions of rHA the intensity of resonance n‘ is reduced with an equal increase in intensity of resonance n (Fig. 1, original peak height ratio n: n’ = 0.2:O.S). The changes in relative inten- sities of peaks n and n’ are shown in Fig. 2. Titration of rHA with Et,PAuCI results in the complete disappearance of peak n’ after addition of 0.5 mol/mol. Addition of 0.3 mol NaSGlc/mol to the final Et,PAuCl/rHA (0.5 : 1) sample re- sulted in the reappearance of peak n’ with a peak height ratio [n’/(n+n’)] of 0.3. Titration of rHA with 1 mol Et,PAuSGI- cAcdmol reduced the peak height ratio [n’/(n+n’)] from 0.8 to 0.5 (Fig. 2).

365

n

\ 9

n' /

I ' l 8.2 8.0

PP"

n \

n \

4

. n

.il-. r - lm

8.2 8.0 8.2 8.0

Fig. 1. The effect of Et3PAuCl and NaSGlc on the His Hcl reso- nances of rHA. 600-MHz 'H-NMR spectra of (A) rHA pH* 6.4, (B) rHA + 0.24 mol Et,PAuCYmol, (C) rHA + 0.5 mol Et,PAuCY mol, (D) rHA + 0.5 rnol Et,PAuCVmol + 0.3 mol NaSGlc/mol. Peaks n and n' are assignable to He1 of His 3 in two different envi- ronments.

1

0.8 - A -

C + c 0.6 - =- C - 0

v

.- c

0.4 - E m - .- a, f

a, a -

0 -

5 0.2 -

0 0.2 0.4 0.6 0.8 1

Gold compound added (mollmol rHA)

Fig.2. The effect of gold complexes on peaks n and n' of rHA (assigned to His3 imidazole Hcl). The peak height of n' as a frac- tion of peak height n + n' is plotted versus rnol (0) Et,PAuSGlcAc, and (0) Et,PAuCI addedmol rHA. Addition of thiols alone had no effect on these rHA resonances.

As with rHA, on addition of Et,PAuSGlcAc,, Et,- PAuSGlc or Et3PAuC1 to solutions of BSA the intensity of resonance n' was reduced with an equal increase in intensity of resonance n (Fig. 3). The change in relative intensity of peaks n and n' on titration of BSA with these complexes is shown graphically in Fig. 4. Titration with Et,PAuCl appears to result in complete reduction of resonance n' after addition

7-1 I I I

7.9 7.6 4.0 3.5 3.0 6 ( P P ~ )

Fig. 3. The effect of gold complexes on the 'H-NMR spectrum of BSA. 500-MHz spectra of (A) BSA pH* 7, (B) BSA + 0.92mol Et,PAuSGlc/mol. (C) BSA + 1.05 mol Et,PAuSGlcAcdmol, (D) BSA + 0.5 mol Et,PAuCVmol, Assignments: (a) Cys resonances from m S G l c ; (b) SGlc resonances from m G l c ; (c) m y s ; (g) glycerol impurity; (n , n') His3 of BSA.

Thiols n + n'

Gold Complexes n'+ n

0 0.2 0.4 0.6 0.8 1

Compound added (mol/mol BSA)

Fig. 4. The effect of gold complexes and thiols on peaks n and n' of BSA (assigned to His3 imidazole Hcl). The peak height of n' as a fraction of peak height n + n' is plotted versus moVmol BSA of added NaSGlc (0), HSGlcAc, (U), dithiothreitol (O), glutathione (A). Et-PAuSGlc (0). Et,PAuSGlcAc, (W) and Et,PAuCI (X). of about 0.4 mol/mol, whereas titration with Et,PAuSGlcAc, \ ~ , , ~~~~

366

n n‘

n n L n n’

n n‘

I’

w & M 1 n n’

‘I

l-----l I I I

719 7.’6 410 3’. 5 3.0 6 (PPW

Fig. 5. The effect of thiols on the ‘H-NMR spectrum of BSA. 500- MHz spectra of (A) BSA pH* 7, (B) BSA + 0.5 mol NaSGlc/mol, (C) BSA + 0.5 rnol HSGlcAcJmol, (D) BSA + 0.5 mol dithiothrei- tol/mol, (E) BSA + 0.5 mol glutathione/mol. Assignments: (a) Cys resonances from m G l c ; (b) SGlc resonances from C G l c ; (c) m y s ; (d) glutathione resonances from Glu(-&ys; (e) Cys resonances from ~ i ~ ( - ( 2 y ~ - ~ i ~ ) Cys; (g) glycerol impurity; (n, n’) His3 of BSA.

or Et,PAuSGlc requires about 1 mol gold complexlmol to complete the conversion of peak n’ into peak n.

Changes in the 2.5-4.3-ppm region of the ‘H-NMR spectrum of BSA (Fig. 3) were also observed with addition of Et,PAuSGlcAc, and Et,PAuSGlc but not with Et,PAuCl. On addition of Et,PAuSGlcAc,, resonances assignable to m y s are observed, whilst on addition of Et,PAuSGlc res- onances assignable to the mixed disulphide m G l c are seen. The assignments were made by comparison with stan- dard spectra of these compounds.

Thiols The addition of a range of thiols (NaSGlc, HSGlcAc,,

glutathione, dithiothreitol) to BSA also had effects on the ‘H- NMR spectra as shown in Fig. 5. In the His HEI region each

the intensity of peak n‘, the opposite of the relative intensity change observed with the gold(1)phosphine complexes. The intensity changes on titration of BSA with the thiols are shown in Fig. 4; all plateau at a ratio peak n‘lpeak n of 0.7 : 0.3; after addition of ~ 0 . 4 mol thiollmol. In the 2.5- 4.3-ppm regions of the ‘H-NMR spectra (Fig. 5 ) it can be seen that addition of NaSGlc or glutathione gave rise to reso- nances assi nable to the mixed disulphide species m G l c

thiothreitol produced resonances assignable to m y s . Again, assignments were made by comparison with standard spectra.

In contrast, addition of NaSGlc (up to 0.25 mol/mol) to rHA had no effect on the resonances for His3 ; resonances for unreacted NaSGlc were observed in the aliphatic region of the spectrum (not shown).

During the course of this work it was also noted that the relative intensity of peaks n and n’ in the crystalline sample of BSA stored at 4°C changed with time from an original nln‘ ratio of 0.5 : 0.5 to 0.6 : 0.4 over a period of 18 months.

and Glu(- 8-7 ys-Gly) ys, whilst addition of HSGlcAc, or di-

DISCUSSION

Several studies of the interaction of gold complexes with albumin have been reported previously. On addition of the anti-arthritic drug auranofin to BSA, triethylphosphine gold(1) binds at Cys34 to produce the species Alb-‘Cys’34- SAuPEt, and an equivalent amount of the -SGlcAc, ligand is displaced [ 13 - 151. Reaction with Et,PAuCl also produces Alb- ‘Cys’34-SAuPEt3 in an amount not exceeding the thio- late content of the BSA. Excess Et,PAuCl appears to bind predominantly at weak sites with nitrogen ligands (possibly His residues) and as the p-thiolato complex Alb-‘Cys’34- S(Au-PEt,), [14]. However the latter complex only forms in significant amounts beyond BSA/Et,PAuCl molar ratios of 1 : 1.3 and was therefore not detected in the present experi- ment. Addition of either Et,PAuCl or auranofin to BSA artifi- cially blocked with Cys to give the disulphide Alb- ‘Cys’34- S C y s (i.e. thiolate content of zero) results in no production of A1b-‘Cys’34-SAuPEt3 or Alb-‘Cys’34-S(Au-PEt3), [14].

It has also been reported that decreases in thiolate content of some BSA samples on storing does not prevent reaction with auranofin [14]. This has been explained as an oxidation of the free thiolate to a sulphenic acid, i.e. Alb-‘Cys’34- SO,H, which is reduced by the thiolate ligand released on reaction of auranofin with the free thiolate form of albumin

BSA has often been preferred to HSA for work on gold complexation since commercial samples are apparently less heterogeneous than those of HSA, especially with respect to the state of Cys34. The sequence identity between HSA and BSA is high (80% [l]), in particular Cys34 is conserved, and results obtained for BSA have been expected to be relevant for HSA. In this work we have used rHA of a high state of purity and BSA.

The results reported here provide further evidence for re- actions of Au(1) phosphine complexes with Cys34, and also demonstrate that they are accompanied by previously unde- tected changes in the structure of albumin. The most notable new finding is the sensitivity of His3 to reactions occurring at Cys34. His3 apparently exists in two different structural states (n and a’), which give rise to close, but distinct, His Hcl resonances 1101 (slow exchange on the NMR time

(Alb-‘Cys’34-S-).

thiol reduced the intensity of peak n and increased equally scale). In the original samples of BSA and rHA used in this

3 67

study the population ratios (nln’) were near to 0.5 : 0.5 and 0.2 : 0.8, respectively, with n’ corresponding closely to the SH contents of the albumins (-0.5 rnol SH/mol BSA and ~ 0 . 8 mol SHlmol rHA).

Reaction of Et,PAuCl with rHA reduces the amount of peak n‘ and increases that of peak n by an amount propor- tional to the added Et,PAuCl. It is clear that the free thiolate form of the protein gives rise to the n’ resonance for His3 and that on binding Et,PAu+ at Cys34 a change in the structure of the protein occurs affecting the environment of His3 and giv- ing rise to peak n. This can be represented by the following equation :

rHis3(n)

Alb-‘Cy~’34-S- + Et,PAuCl * A I ~ - ‘ C ~ S ’ ~ ~ - S A L I P E ~ , + C1-, LHis3(n‘)

A change in structure is supported by effects on resonances in other regions of the spectrum on binding Et,PAu+, e.g. the new resonance in the high-field region is clear evidence of a change in structure as this must arise from, for example, a methyl group being brought into close proximity with the face of an aromatic ring. The effects on other resonances assignable to His He1 protons also indicate a change in struc- ture as their pKa values or limiting chemical shifts have been perturbed.

Addition of NaSGlc to a sample of rHA/Et,PAuCl (1 .O : 0.5) caused the reappearance of peak n’ suggesting that thioglucose (-SGlc) competes effectively with Alb- ‘Cys’34- S- for Et,PAu+ via a ligand exchange process:

,-His3(n)

Alb-‘Cys’34-SAuPEt, + -SGk Alb-‘Cys’34-S- + Et,PAuSGlc. LHis3(n‘)

This reaction explains why, on addition of Et,PAuSGlcAc, to rHA, the decrease in peak n’ is not stoichiometric, as the displaced thiosugar competes with Cys34 of albumin for the Et,PAu+, i.e under the conditions used here the equilibrium constant for the reaction below is approximately 1.

p i s 3 ( n )

Alb-‘Cy~’34-S- + Et,PAuSGlcAc, * Alb-‘Cy~’34-SAuPEt, + -SGlcAc, LHis3 (n‘)

Addition of thiols to samples of BSA caused an increase in peak n’ and the release of disulphides containing cysteine, i.e the reaction below is occurring:

Alb-Tys’34-Wys + RSH == Alb-‘Cys’34-S- + R-ys

rHis3(n’)

LHis3(n‘)

This accounts for the difference in the reactions of Et,. PAuSGlcAc, with rHA and BSA. On addition of Et,PAuSGl- cAc4 to BSA some of the released thiosugar reacts with Cys blocked BSA (Alb- ‘Cys’34-sys) and is therefore not avail- able to compete with Cys34 of BSA for Et,PAu+. The final low-molecular-mass disulphide products arising from BSA depend on the relative redox potentials of the thiols and dis- ulphides potentially formed, and are either mixed disulphides of Cys with the added thiol, or cystine formed via disulphide interchange reactions.

On storage of crystalline BSA at 4°C the ratio of peaks n and n’ changed gradually, increasing the amount of peak n. It has previously been suggested that an oxidised Cys34 form of the protein (Alb-‘Cys’34-SOXH) is produced on storage

[14] and it would appear that this form of the protein has His3 in the same environment as the disulphide- and gold- blocked forms, and gives rise to peak n. This species appears to be reduced on addition of thiols. However, the sample of bovine albumin used in this study also contains approxi- mately 30% of a species in the His3(n) form which is not reduced by addition of thiols. The nature of this species will be the subject of further study.

It is clear that a structural transition occurs in albumin which is dependent on the environment of Cys34. When Cys34 is in the reduced, free-thiolate form the structure of the protein is such that His3 gives rise to peak n’. When Cys34 is oxidised (as a disulphide or sulphenic acid) or blocked by Et,PAu+, a structural transition occurs affecting the environment of His3 thus giving rise to peak n. The thio- late form of the Cys34 side chain is probably crucial to the stabilisation of the n’ form due to salt bridge formation in its local pocket. All of the Cys34-modified forms remove the potential for such salt bridging (but not hydrogen bonding). The size of the blocking group is not critical in destabilising the n’ form with respect to the n form. Peaks n and n’ of His3 coalesce below about pH6, for both BSA and rHA, implying a similar associated ply, for the SH group of Cys34, a value close to that determined previously (pK,<5) by Lewis et al. [4].

A view of the cleft containing Cys34 from the X-ray crystal structure of human albumin [161 reveals Cys34 and Pro35 as the last two residues in the loop connecting helices 2 and 3 of domain I. The side-chain of Cys34 is buried inside the cleft and close to His39 such that a salt bridge could easily form and may stabilise the buried form of the protein. Blocking of Cys34 by Et,PAu+ or oxidation would prevent such stabilisation, and favour the exposed form.

The movement of the side-chain of Cys34 to an exposed position on binding Et,PAu+ is strongly supported by the observation of well-resolved resonances for the ethyl groups of Et,PAu+ bound to albumin. It is very unlikely that such sharp resonances would be observed for Et,PAu+ bound to Cys34 in the orientation shown in the X-ray structure [3, 161 as the ethyl groups would lie deep inside the binding pocket, have a very low mobility, and give rise to broad (unobserv- able) resonances.

Cis-trans isomerisation of Pro35 could mediate move- ment of the inter-helical loop to the N-terminal side, rotating the side-chain of Cys34 from a buried (n’) to an exposed (n) environment. A small rearrangement of helices 1 and 2 with respect to the rest of the domain could transmit this effect to the N-terminus, so affecting the environment of His3. It is not clear whether Et,PAu+ is able to penetrate the binding pocket to attack the free thiolate n’ form of the protein. It is possible that the free thiolate form of the protein exists in equilibrium between the buried (n’) and exposed (n) forms with the equilibrium being strongly in favour of the buried form. The equilibrium would then allow reactions to occur with the exposed thiolate form of the protein forcing the equilibrium progressively towards the exposed form, e.g. :

rHis3(n) rHis3(n) Alb-‘Cys’34-S- + Et3PAuCl == Alb-‘Cys’34-SAuPEt, + C1-

JI Alb-‘Cys’34-S-

LHis3(n‘) The existence of such an equilibrium may explain the obser- vation that addition of 0.5 mol Et,PAuCI to 1 mol of a sample of rHA (with a thiolate content of 0.8 mol/mol) is sufficient

368

to complete the conversion of peak n’ into peak n (original ratio nln’ = 0.2:0.8), if the exchange of Et,PAu’ between Alb-‘Cys’34-S- molecules is fast relative to the rate at which His3(n)-Alb-‘Cys’34-S- converts into His3(n’)-Alb-

The structural transition of albumin proposed here may explain several previously reported observations and lead to some important conclusions. For example, it may provide a trigger for proteolytic degradation, since enzymes such as cathepsins are unable to attack mercaptalbumin but degrade blocked forms [17]. Feedback between the binding of anti- arthritic gold drugs to Cys34 and the environment of His3 [the locus of the strong Cu(I1) binding site of albumin] may be related to the known relationship of copper homeostasis, albumin levels and the disease state of rheumatoid arthritic patients. We are currently investigating the interdependence of Au(1) and other metals binding to albumin. The competi- tion of thiols for Et,PAu’ bound to albumin suggests that thiol levels in serum may be critical to the bioavailability of gold drugs by controlling the extent of binding to serum albumin.

In X-ray crystallisation studies of HSA and rHA, no use of gold(1) as a heavy atom derivative has been reported [2, 31. We anticipate that attempts to prepare gold derivatives by soaking crystals of albumin in solutions of Et,PAuSGlcAc, may prove unsuccessful. The crystalline protein may be un- able to undergo the structural transition to the exposed (n) form and thus accommodate the Et3PAu+, or disordering due to the structural transition may result. Alternatively, if cocrystallisation with Et,PAuSGlcAc, is attempted, compli- cated equilibria are likely to occur which are highly depen- dent on the microheterogeneity at Cys34. Our results suggest that the use of Et,PAuCI may be more successful, although problems with eventual loss of Et,PO via reduction of albu- min disulphide bonds must also be considered 1181. It is pos- sible that the use of gold(1) thiolate complexes such as auro- thiomalate may produce gold-albumin complexes more sui- table for X-ray studies and we are investigating the effect of the binding of such complexes on the structural transition described here.

‘Cys’34-S-.

CONCLUSIONS Gold(1) phosphine binding, disulphide blocking and oxi-

dation of Cys34 of albumin appear to have similar effects on the shift of the Hcl ‘H-NMR resonance of His3. Such a communication between His3 and Cys34 may be explained by a structural transition involving small changes in the ar- rangement of intervening helices 1 and 2 of domain I. This may be mediated by a cis-trans isomerisation of Pro35, changing the environment of Cys34 from a buried to an ex- posed one.

The reduction of cysteine-blocked forms of BSA by thiols is concluded from the appearance of c ’ y s y s or m R products. No products which would imply the exis- tence of glutathione-blocked forms of the protein are ob- served.

The structural transition may have important conse- quences in signalling degradation of albumin and in copper homeostasis in rheumatoid arthritis patients. Characterisation of gold-albumin in the crystalline state (e.g. as a heavy atom derivative) will probably require crystallisation of a gold- albumin complex prepared under well defined and carefully controlled conditions.

We thank the Wellcome Trust, the Medical Research Council, the Science and Engineering Research Council and Delta Biotech- nology for their support for this work. We thank Dr D. C. Carter and Dr X-M He for providing views of the X-ray crystal structure of human albumin prior to publication and are grateful to the Bio- medical NMR Centre Mill Hill for the provision of NMR facilities.

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W. Putnam, ed.), 2nd edn., vol. I, pp. 133-181, Academic Press, New York.

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3. Carter, D. C. & He, X-M. (1992) Atomic structure and chemis- try of human serum albumin, Nature 358, 209-215.

4. Lewis, S. D., Misra, D. C. & Shafer, J. A. (1980) Determination of interactive thiol ionisations in bovine serum albumin, gluta- thione and other thiols by potentiometric difference titration, Biochemistry 19, 6129-6137.

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