From the Reactions of cisplatin
(cis-[PtCl2(NH3)2])
with albumin are thought to play an important role in the metabolism of
this anticancer drug. They are investigated here via (i) labeling of
cisplatin with 15N and use of two-dimensional
1H,15N NMR spectroscopy, (ii) comparison of
natural human serum albumin with recombinant human albumin (higher
homogeneity and SH content), (iii) chemical modification of Cys, Met,
and His residues, (iv) reactions of bound platinum with thiourea, and
(v) gel filtration chromatography. In contrast to previous reports, it
is shown that the major sulfur-containing binding site involves Met and
not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Additional monofunctional adducts involving other Met residues and
Cys-34 are also observed. During the later stages of reactions of
cisplatin with albumin, release of NH3 occurs due to the
strong trans influence of Met sulfur, which weakens the
Pt-NH3 bonds, and protein cross-linking is observed. The
consequences of these findings for the biological activity of
cisplatin-albumin complexes are discussed.
Cisplatin,
cis-[PtCl2(NH3)2], is
extensively used for the treatment of testicular and ovarian cancers
and increasingly against other types of solid tumors (head/neck, lung,
cervical, and bladder) (1-4). The cytotoxic effect of cisplatin is
thought to be due to attack on DNA bases and induction of apoptosis in
cancer cells (5, 6). It is also able to bind to a number of extra- and intracellular proteins (7-11). Inactivation of certain enzymes due to
cisplatin binding is likely to be responsible for its appreciable side
effects, mainly nephrotoxicity and ototoxicity (12-14).
In body fluids, cisplatin is readily attacked by nucleophiles with
exchange of one or both chloride ligands to form high and low molecular
mass complexes (15, 16). One day after rapid intravenous infusion of
cisplatin, 65-98% of platinum in blood plasma is protein-bound
(17-19), while no unbound platinum has been detected at any time in
blood plasma of patients after slow 20-h infusions (18). Protein
binding results in significantly lower urinary excretion and an
increased tissue deposition of platinum (18).
Reaction between cisplatin and serum albumin is thought to be the main
route for platinum binding in human blood plasma (16). Cisplatin
binding to albumin is essentially irreversible with less than 5% loss
of protein-bound platinum after extensive dialysis (15, 20).
Human serum albumin (HSA)1 is
a single-chain 66-kDa protein, which is largely Although it is widely thought that albumin-bound platinum is
therapeutically inactive (19, 24, 25), several clinical and
experimental observations are in conflict with this opinion. (i)
Hypoalbuminemic patients respond poorly to cisplatin treatment (26-28), (ii) the infusion of preformed cisplatin-albumin complexes significantly increases patient survival times (29, 30), and (iii)
HSA-cisplatin complexes are cytotoxic to carcinoma cells (29, 31).
Thus, the antitumor activity of infused cisplatin may be determined by
both free and albumin-bound drug. Additionally, albumin binding may
prevent some of the side effects of cisplatin treatment, especially its
nephrotoxicity (30, 32). Therefore, an understanding of the molecular
mechanism of albumin-cisplatin interactions may have an impact on the
optimization of strategies for cisplatin treatment.
Despite numerous studies of cisplatin-albumin binding during the last
few decades (7, 15, 18, 19, 33-37), the precise mechanism of
interaction is still poorly understood. Gonias and Pizzo reported that
carboxyamidomethylation of the SH group in bovine and human serum
albumin resulted in a 4-5-fold decrease in platinum binding and
suggested formation of a single monodentate complex of cisplatin with
the free sulfhydryl group (Cys-34) (7, 33). Bovine non-mercaptalbumin,
obtained via reaction with cystine, also had a low affinity for
cisplatin, compared with mercaptalbumin (38). However, the
disulfide-type dimers of BSA with no free SH groups still bound
substantial amounts of cisplatin (38). In addition, blocking the free
SH group has been reported to have no effect on the binding of 2 mol eq
of transplatin to HSA (7). These data suggest the existence of
additional platinum binding sites on albumin. Momburg et al.
(34) also suggested the existence of two albumin binding sites for
cisplatin, but apart from Cys-34, the nature of the other cisplatin
binding site was not elucidated.
The aim of the present work was to study the reaction of cisplatin with
intact and chemically modified recombinant human albumin (rHA), and
with HSA using one-dimensional 1H and two-dimensional
1H,15N HSQC NMR spectroscopy to characterize
platination sites under different experimental conditions.
Two-dimensional 1H,15N HSQC NMR spectroscopy
has proved to be extremely powerful in elucidating the chemistry and
biochemistry of 15N-labeled platinum ammine complexes. The
15N chemical shift of the ammine is diagnostic of the
ligand coordinated to platinum in the trans-position (39).
Recent studies have included those on the interaction of platinum drugs
with nucleic acids, amino acids, and peptides (39-41).
The experiments reported here reveal for the first time that strong
binding sites other than Cys-34 play a major role in albumin-cisplatin interactions in solution.
Materials--
cis-[PtCl2(15NH3)2]
and
cis-[Pt(15NH3)2(H2O)2]2+
were prepared according to literature procedures (42). Iodoacetamide,
N-ethylmaleimide, 5,5'-dithio-bis(2-nitrobenzoic acid),
Norit-A (Sigma), iodomethane (Merck), diethylpyrocarbonate
(Acros-Organic), DEAE-Sepharose Fast Flow, SP-Sepharose Fast Flow
(Amersham Pharmacia Biotech), and polyethylene glycol 4000 (Fluka) were
used as received. All other chemicals were of the highest quality
available from Sigma and Aldrich.
Albumin Samples--
rHA was prepared by Delta Biotechnology Ltd
(batches GA950202 and R970103). Samples of rHA were extensively
dialyzed against 100 mM ammonium bicarbonate, pH 7.9, and
freeze-dried. HSA was obtained from the Scottish National Blood
Transfusion Service (batches HA(20)60440 and SPPS (P-3-671)). Crude HSA
was precipitated with 25% polyethylene glycol, purified by sequential
chromatography on DEAE-Sepharose and SP-Sepharose Fast Flow (43), and
defatted by activated charcoal (44). Purified samples were dialyzed
against 100 mM ammonium bicarbonate and freeze-dried.
rHA Chemical Modification--
Cys-34-blocked rHA was prepared
by incubation of albumin solution (40-60 mg ml Department of Chemistry,
Delta Biotechnology Ltd,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helical and
consists of three structurally homologous domains, organized into a
heart shape (21). HSA contains 17 disulfide bridges and one free thiol
at Cys-34. Albumin is known to bind to various endogenous metabolites,
metal ions, and drugs (21, 22). Binding of drugs to serum albumin
affects their metabolism, efficacy, and body distribution (23).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1) in 100 mM ammonium bicarbonate, pH 7.9, with 2 mol eq of
iodoacetamide (45) or N-ethylmaleimide (46) for 12-16 h at
ambient temperature in the absence of light. Excess of the blocking
agent was removed by dialysis against the same buffer.
1) in 0.1 M citric acid-phosphate
buffer, pH 4.0. The two-phase solution was stirred at ambient
temperature for 20 h in the dark. At the end of this period,
iodomethane was removed by dialysis against 0.2% w/v NaN3
and then water.
Preparation of rHA-Cisplatin Complexes-- In most experiments, cis-[PtCl2(15NH3)2] was freshly dissolved in 10 mM phosphate, 100 mM KCl, to a concentration of 1 mM. Freeze-dried albumin was added to an equivalent concentration, followed by the addition of deuterium oxide to 10% to provide an NMR lock signal. The final pH of the solution was adjusted to 6.4 using small aliquots of 2.0 M NaOH. In several experiments, rHA was dissolved to a final concentration of 1 mM in buffer solutions containing 0.5-2 mM cisplatin.
Determination of Free SH Groups--
The free SH content of
albumin was determined after 24 h of incubation with cisplatin.
Free cisplatin was removed by ultrafiltration with a Centricon-10
concentrator (Amicon, Beverly, MA). The thiol contents of rHA and HSA
were estimated using Ellman's reagent, 5,5'-dithio-bis(2-nitrobenzoic
acid) (49, 50). The amount of generated p-nitrothiolate
anion was calculated using the extinction coefficient 13,600 M1 cm
1 at 412 nm (49). The
concentration of albumin was measured using the absorption at 279 nm
assuming A279 (1 mg ml
1, 1 cm) = 0.556 (51).
FPLC--
Gel filtration of cisplatin-albumin complexes was
carried out on a Superdex 200 HR column (internal diameter 1 cm; length 30 cm) with an FPLC system (Amersham Pharmacia Biotech) at ambient temperature. Albumin samples were diluted to a concentration of 5 mg
ml1 and 200 µl of the protein solution was loaded onto
the column. Elution conditions were 50 mM sodium phosphate,
100 mM NaCl, pH 7.0, with flow rate of 0.5 ml
min
1 and detection at 280 nm.
NMR Spectroscopy--
One-dimensional 1H and
two-dimensional 1H,15N HSQC NMR spectra were
acquired at 500.13 MHz using a Bruker DMX500 NMR spectrometer equipped
with a tunable triple resonance (TBI) probehead
[1H,13C,X] incorporating an
actively shielded z-field gradient coil. One-dimensional
1H NMR spectra were acquired using a
double-pulsed-field-gradient spin-echo pulse scheme (52), based on the
WATERGATE sequence, namely
90°x-Gza-sel90°x-180°x-sel90°
x-Gza-Gzb-sel90°
x-180°x-sel90°
x-Gzb-ACQ.
Typically 32 transients were acquired into 20,000 complex data points
using digital quadrature detection over a frequency width of 10 kHz
with a recycle delay of 1.4 s. Solvent-selective 90° pulses
(sel90°) were of a rectangular profile with a pulse
duration of 2.3 ms. Sine-shaped z-gradient pulses of 2 ms
duration were followed by field-gradient-recovery delays of 100 µs.
Gradient strengths of 5.5% and 3.0% were used for gradients "a"
and "b," respectively. Data were zero-filled to 32,000 data points
and apodized using an optimal combination of an exponential
line-broadening function of 2 Hz and an unshifted sine-bell function
(53) prior to Fourier transformation. All spectra were recorded at 310 K.
NMR Peak Integration-- All two-dimensional HSQC data acquired at 500 MHz were processed using the software XWINNMR version 1.3 (Bruker Spectrospin Ltd.). Prior to volume integration, flat baseplanes were established for all data sets by applying a baseplane correction. Two types of region were chosen for integration. (i) The overall intensity of each spectrum of protein-bound cisplatin was measured by integrating the entire two-dimensional region where signals were observed, and (ii) the volume integral of the 1H-15N signal from the starting material was measured excluding the areas in which 195Pt satellites occur.
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RESULTS |
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Reactions of cisplatin with both recombinant human albumin and
human albumin isolated from blood serum were studied. Although rHA and
HSA are similar in amino acid composition, secondary structure and
globular packing, they differ in two respects, namely in free thiol
content (approximately 0.9 mol SH mol1 rHA and 0.4-0.5
mol SH mol
1 HSA) and in the greater structural
heterogeneity of HSA compared with rHA (55). Cisplatin-albumin
interactions were studied mainly at pH 6.4, where albumin exists
predominantly as one structural isomer, namely the N-form (22). Several
experiments were also carried out at pH 7.4 and 5.0. In most
experiments, KCl was added to suppress cisplatin hydrolysis (as would
be the case under extracellular conditions).
NMR Study of the Reaction of Cisplatin with Intact Human Albumin in Media with Added Chloride
One-dimensional 1H NMR-- One mol eq of cisplatin added to an albumin solution (1 mM rHA in 10 mM phosphate and 100 mM chloride, pH 6.4) induced changes in the protein one-dimensional 1H NMR spectrum. Specifically, peaks at 2.19 ppm and 2.05 ppm significantly decreased in intensity relative to other peaks in the aliphatic region over a 17 h period at 310 K (data not shown). No other significant changes were observed. There was also a decrease in the overall signal-to-noise ratio, which was significant after 17 h of reaction.
Two-dimensional 1H,15N HSQC NMR-- Fig. 1 shows two-dimensional 1H,15N HSQC NMR spectra of solutions of 1 mM [15N]cisplatin with rHA (A) and fatty-acid-free HSA (B) in a 1:1 molar ratio recorded after a 13-h incubation in 10 mM phosphate buffer, pH 6.4, with 100 mM KCl present at 310 K. The spectrum of [15N]cisplatin-rHA complex contains a cross-peak due to cis-[PtCl2(15NH3)2] (labeled as Pt) and eight cross-peaks attributable to cisplatin-albumin adducts (labeled as a, a', b, c, d, d', e, and f). The spectrum for the [15N]cisplatin-HSA reaction is very similar to that for rHA, and differs only in the higher intensity of the cross-peak from unreacted cisplatin, and in the negligible intensity of cross-peak d' (Fig. 1B). 15N and 1H chemical shifts of observed cross-peaks are listed in Table I.
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NMR Study of the Reaction of Cisplatin with Intact rHA in Media without Added Chloride
Two-dimensional 1H,15N HSQC NMR-- The reaction of rHA with [15N]cisplatin (1 mM, 1:1) was also studied in 10 mM phosphate buffer pH 6.4 without added chloride. In the early stages of this reaction, the two-dimensional 1H,15N HSQC NMR spectra contained the same cross-peaks as described for rHA-[15N]cisplatin in the presence of 100 mM chloride (Fig. 5, A and B). However, cross-peaks d and e/f had completely disappeared from the spectrum after 17 h of reaction (Fig. 5C).
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Effect of Cisplatin on the Free Thiol Content and Heterogeneity of rHA
SH Group Determination--
The free SH content of two batches of
rHA and two of HSA before and after reaction with cisplatin was
determined by the DTNB-method. rHA samples contained 0.78 and 0.75 mol
of free thiol/mol of protein, while for HSA samples the amount of free
SH was significantly lower, 0.49 and 0.29 mol mol1 (Table
II). After 24 h of reaction with an
equimolar concentration of cisplatin, the SH content of the two rHA
samples decreased similarly, from 0.78 to 0.69 and from 0.75 to 0.67 mol mol
1. Incubation of rHA (1 mM) with a
2-fold molar excess of cisplatin resulted in a more pronounced decrease
of free SH, from 0.75 to 0.54 mol mol
1. Reaction of HSA
(1 mM) either with an equimolar concentration or with a
2-fold molar excess of cisplatin did not change the SH content of
either batch of HSA. Additionally, albumin-bound cisplatin had no
influence on the reaction of DTNB with SH-blocked rHA (Table II).
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FPLC Data-- Fig. 7 shows typical gel-filtration chromatograms of control rHA and its complexes with cisplatin. The chromatogram of control rHA consisted of one strong peak with a retention time of 28.6 min (91% calculated by absorbance at 280 nm) due to rHA monomer and a small peak at 25.1 min due to albumin dimers. The chromatogram of the 1:1 cisplatin-albumin reaction (1 mM, reacted 24 h in 10 mM phosphate, 100 mM KCl, pH 6.4) was significantly different from the control; the intensity of the monomer peak decreased to 58% of the total, that of the dimer peak increased, and a new peak with a retention time of 23.3 min appeared (Fig. 7B). After reaction of rHA with a 2-fold molar excess of cisplatin under similar conditions, only 43% of the monomer form remained, the peak at 23.3 min was more intense, and a new broad peak with a retention time of ~20 min was present (Fig. 7C). Incubation of cisplatin with 1 mol eq of SH-blocked (via reaction with iodoacetamide) rHA under similar conditions, led to a smaller decrease in the amount of monomeric albumin: from 91% to 70%.
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NMR Study of the Reaction of Cisplatin with Chemically Modified rHA in Media with Chloride
SH-blocked rHA-- On reaction of Cys-34 blocked rHA (iodoacetamide) with cis-[PtCl2(15NH3)2], the 1H,15N cross-peaks a, b, c, d, and e/f were still observed in the two-dimensional 1H,15N HSQC NMR spectrum at various incubation times up to 17 h (Fig. 8). The main difference between spectra from the reactions of blocked and unblocked albumin was the absence of cross-peaks a' and d' in spectra of SH-blocked rHA. Additionally, in the two-dimensional 1H,15N HSQC NMR spectrum of SH-blocked rHA, the cross-peak for unbound cisplatin (peak Pt, Fig. 8) was still visible after a reaction time of 17 h, while this signal disappeared from the spectrum of unblocked albumin after 13 h incubation with cisplatin under similar conditions. Exactly the same changes in the two-dimensional 1H,15N HSQC NMR spectra were obtained for the reaction of cisplatin with N-ethylmaleimide-modified albumin (data not shown).
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Methionine-modified rHA--
The methionine residues of rHA were
methylated by a 500-fold molar excess of iodomethane at pH 4.0. The
reaction with iodomethane resulted in suppression of the singlet
resonances assignable to methionine S-CH3 groups in the
region 1.9-2.3 ppm of the 1H NMR spectrum (data not
shown). Fig. 9A shows the
two-dimensional 1H,15N HSQC NMR spectrum
obtained after 13 h of the reaction of
[15N]cisplatin with methionine-modified rHA (1:1, 1 mM, in the presence of chloride, pH 6.4). The intense
signals in this spectrum can be assigned to free cisplatin and its
partially hydrolyzed products. Only a very weak cross-peak
e/f was observed in the ammine trans to
nitrogen/chloride region together with a weak signal at 46.2/4.13 ppm
in the ammine trans to sulfur region, probably attributable
to cross-peak c with a low frequency 1H shift.
The above cross-peaks were clearly visible in the spectrum after only
9 h and retained weak intensities up to 17 h of reaction (data not shown).
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Histidine-modified rHA-- Histidine residues of rHA were modified by reaction with diethylpyrocarbonate in 0.1 M phosphate buffer pH 6.0. This procedure resulted in modification of 6 His imidazole rings, as calculated from the absorption at 240 nm of the N-carbethoxyimidazole formed. Histidine modification produced substantial changes in the 1H NMR spectrum of rHA, in particular, suppression of the six most intense (sharp) resonances in the aromatic region of the spectrum (data not shown). The two-dimensional 1H,15N HSQC NMR spectrum of histidine-modified rHA after 13 h of reaction with [15N]cisplatin contained the same cross-peaks a, b, c, d, and e/f as the spectrum of unmodified albumin (Fig. 9B). The differences concerned a slight decrease in the relative intensities of cross-peaks d, e/f, and c, and a high frequency 1H shift of peak e/f in the spectrum of histidine-modified rHA.
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DISCUSSION |
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The biotransformation of cisplatin occurs directly in biological fluids via the formation of complexes with plasma proteins, especially serum albumin (15, 16, 18-20). Cisplatin-albumin adducts may play an important role in determining the body distribution of platinum, and in reducing platinum nephrotoxicity and ototoxicity (32, 56). However, there were only a few studies of the fate of albumin-bound platinum, and its role, if any, in the antitumor activity is controversial.
Takahashi et al. (57) and Hoshino et al. (31) have reported that cytotoxic effects occur only at very high concentrations of cisplatin-albumin and concluded that the complex was unlikely to contribute to the antitumor activity of cisplatin. A short report by DeSimone et al. (29), however, claimed that a complex of cisplatin and albumin possessed an equivalent antitumor activity to free cisplatin in seven transplantable animal tumor models. They also obtained several responses from patients with various types of tumor using a cisplatin-albumin complex. Holding et al. (30) noted the effectiveness of cisplatin-albumin complexes in the treatment of patients with squamous cell carcinoma of the head and neck. An interesting observation in this study concerned the tumor concentration of platinum, which was considerably higher after cisplatin-albumin administration than after conventional cisplatin therapy.
The reported contradictions concerning the antitumor activity of cisplatin-albumin complexes may arise from the use of different methods for the preparation of cisplatin-albumin complexes. Understanding the exact mechanism of cisplatin-albumin binding may enable improved cytotoxic formulations to be developed.
Two-dimensional 1H,15N HSQC NMR spectroscopy
has proved to be an extremely useful tool in elucidating the chemistry
of 15N-labeled Pt-NH3 complexes (39-41). The
15N chemical shift of the ammine is diagnostic of the
ligand coordinated to platinum in the trans-position (39).
If the ligand trans to ammine is an oxygen-donor ligand,
then the ammine chemical shift falls between 75 and
95 ppm, if
trans to nitrogen/chloride
55 to
70 ppm, and
trans to sulfur between
40 and
50 ppm. Since about 34%
of platinum is 195Pt, which has a spin quantum number
I = 1/2, 1H,15N
Pt-NH3 peaks are 1:4:1 triplets due to 195Pt
spin-spin coupling. The 195Pt satellites are usually
observed only for low Mr platinum complexes. For
larger complexes, such as platinum-albumin adducts, they are broadened
beyond detection due to relaxation via chemical shift anisotropy
(58).
In the current work, reactions of cisplatin with intact and chemically modified rHA and HSA have been studied using one-dimensional 1H and two-dimensional 1H,15N HSQC NMR spectroscopy, together with liquid chromatography, to characterize platination sites under different experimental conditions.
Reaction of Intact Human Albumin with Cisplatin in Media with Chloride
1H NMR--
Reaction of cisplatin with 1 mol eq of rHA
significantly reduced the intensity of singlets at 2.05 and 2.19 ppm
assignable to CH3 groups of methionine residues (59, 60)
in the spectrum of the protein, but formation of cisplatin-albumin
adducts at pH 6.4 did not result in significant suppression of any
signals in the aromatic region of the 1H NMR spectrum. This
region contained six major singlet resonances assignable to imidazole
ring protons of histidines (61). Previous 1H NMR titration
experiments have shown that most of these histidines have
pKa values between 6.5 and 7.5 (61), close to that
of free histidine, and are therefore likely to be exposed to the
solvent.
Two-dimensional 1H,15N HSQC NMR-- Reaction of cisplatin with human albumin was readily monitored by two-dimensional 1H,15N HSQC NMR spectroscopy, both by the appearance of new cross-peaks from drug-albumin adducts and by the disappearance of the cross-peak for free [15N]cisplatin (Figs. 1 and 2).
Eight new cross-peaks attributable to protein-bound drug were observed in reactions of 1 mM rHA or defatted HSA with an equimolar amount of [15N]cisplatin (100 mM KCl, pH 6.4, 310 K) (Fig. 1). Two of these, labeled a' and d', grew in intensity up to 6 h of reaction, but then significantly decreased and had negligible intensities 17 h after mixing (Fig. 2A). Other cross-peaks, labeled a, b, c, d, and e/f, were clearly visible in spectra acquired over the whole course of the reaction (Fig. 2, A and B). There were no differences in the number and the chemical shifts of cross-peaks attributable to protein-bound drug in spectra of rHA and HSA, which implies that these two proteins have identical binding sites for cisplatin (Fig. 1). Additionally, after a 17-h reaction of rHA with an equimolar concentration of cisplatin at pH 7.4, the same cross-peaks were observed as at pH 6.4 (Fig. 3A). It therefore seems likely that the two main structural isomers of albumin (namely the N- and B-forms; Ref. 22), which coexist at physiological pH, form similar adducts with cisplatin. The 1H,15N cross-peak for unbound [15N]cisplatin decreased sharply in intensity with time, and was not detectable in the spectrum after 17 h of reaction (Fig. 2C). Simultaneously the general intensity of the spectrum greatly decreased, being 9-fold less after 17 h than after 2 h of reaction. These results imply that some of the 15N-containing products are not detectable. Different processes may reduce the signal intensities of cisplatin-albumin adducts. First, the loss of 15NH3 ligands of cisplatin may occur during interaction with the protein; second, 1H exchange on 15NH4+ may be too rapid at this pH to allow detection. Additionally, reductions in relaxation times for bound Pt-NH3 may lead to line-broadening and loss of signal intensities (62). The initial growth of cross-peaks a', d', a, c and e/f followed by decay after 6-9 h of reaction (Fig. 2B) suggests the loss of NH3 from bound platinum. Displacement of NH3 has been observed previously in reactions of cisplatin with the sulfur donor of free methionine and cysteine-containing peptides in model solutions (60, 63, 64) and in human blood plasma (65). In the reaction of rHA with [15N]cisplatin, the most intense peaks a and b are likely to represent the major cisplatin-albumin adduct (Fig. 2, A and B). The 15N chemical shift of a is indicative of an ammine ligand trans to a sulfur donor ligand, and that of b, of an ammine trans to nitrogen/chloride, i.e. to species A or B in Scheme I. The other pair of cross-peaks in the ammine trans to sulfur (a') and ammine trans to nitrogen/chloride regions (d'), is likely to represent a second type of cisplatin-albumin complex with a high rate of ammonia loss. Additional cisplatin-albumin adducts give rise to intense cross-peaks in the ammine trans to sulfur (c) and in the ammine trans to nitrogen/chloride regions (d and e/f). It is likely that cross-peak c is paired with both peaks d and e/f, representing two cisplatin-albumin adducts (c-d and c-e/f) with similar sulfur donors and different types/environments of nitrogen/chloride donors. These adducts have different rates of formation and subsequent loss of NH3, which results in variations in their peak intensities at different times of reaction (Figs. 2 and 3).
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Reaction of Intact Human Albumin with Cisplatin without Added Chloride
The assignment of cross-peaks d and e/f to Pt-NH3 trans to chloride ligands in cisplatin-albumin adducts was supported by the investigation of cisplatin-rHA reactions without added chloride (Fig. 5). All the 1H,15N cross-peaks for protein-bound cisplatin observed during the first 9 h of reaction were similar to those observed in the presence of chloride (Fig. 5, A and B). However, more prolonged incubation resulted in the elimination of cross-peaks d and e/f. In this case, these monofunctional cisplatin-albumin adducts would be further modified by hydrolysis with displacement of chloride ligands by aqua/hydroxo ligands.
In addition, rHA was incubated with cis-[Pt(15NH3)2(H2O)2]2+ at pH 5.0 to minimize the formation of hydroxy-bridged dimeric and polymeric platinum species that readily occur above the first pKa value of coordinated H2O (5.37) (69). It is noteworthy that, at this pH, albumin still exists in the native, completely folded N-form (22). Reaction of cis-[Pt(15NH3)2(H2O)2]2+ with equimolar rHA resulted in only the pair of intense cross-peaks a and b for protein-bound drug in the two-dimensional 1H,15N HSQC NMR spectrum (Fig. 6). Other strong peaks were observed in the ammine trans to oxygen region of the spectrum, and probably represent hydroxy-bridged dimeric and polymeric platinum species.
These investigations of the interaction of [15N]cisplatin with rHA under different experimental conditions allow the following types of cisplatin binding sites on albumin to be proposed. (i) The major adduct is a single bifunctional complex of {Pt(NH3)2}2+ involving sulfur and nitrogen donor ligands giving rise to the pair of cross-peaks a and b (e.g. species B in Scheme I); (ii) the second type of site accounts for the two monofunctional adducts giving pairs of signals c-d and c-e/f, involves a sulfur donor of albumin and also contains a chloride ligand, which is apparently rather unreactive perhaps because it is shielded by the protein (species A in Scheme I); (iii) the third type of site is probably also a monofunctional adduct with a sulfur donor from albumin, and appears to undergo rapid loss of platinum-bound NH3 (species C in Scheme I).
Our data show that cisplatin binds to two sites even when albumin is present in a 2-fold molar excess, giving a major bifunctional adduct and a monofunctional one (Fig. 3A). With a 2-fold molar excess of [15N]cisplatin, the proportion of monofunctional adducts significantly increases (Fig. 3B).
The two-dimensional 1H,15N HSQC NMR data suggest that only sulfur and nitrogen/chloride donor ligands of albumin are involved in platinum binding. There are three types of sulfur atom in albumin, namely Cys-34 SH, Met thioether, and cystine disulfide. Although previous work on cisplatin-albumin interactions has suggested that platinum can induce cleavage of disulfide bonds, implying binding of platinum to cystine sulfur (35, 36), a high excess of platinum and very long incubation times were required to achieve this. Therefore, Cys-34 and Met are the likely sulfur ligands for platinum in the present work. From consideration of published x-ray structures of several proteins, it is likely that Pt(II) binds more readily to histidine and the N-terminal amino group (70) than other N donors. However, in our study cisplatin binding did not perturb the resonances for Asp-1 and Ala-2, which were clearly visible in the aliphatic region of the one-dimensional 1H NMR spectrum of rHA. Therefore, the most likely source of a nitrogen donor in the formation of the bifunctional cisplatin-albumin adduct is the imidazole ring of histidine. Pt(II) chelation to S and deprotonated amide N is known for Met peptides and is favored at high pH (71).
To further elucidate the nature of the amino acid residues of albumin that are involved in platinum binding, we studied the free thiol content, aggregation status of rHA, and influence of selective modification of Cys-34, methionines, and histidines on cisplatin-rHA interactions.
Influence of Cisplatin Binding on Free Thiol Content and Heterogeneity of rHA
SH Group Determination-- The reaction of cisplatin with rHA (1:1 molar ratio in the presence of chloride) resulted in a 11-12% decrease in the free SH content for the two batches of rHA investigated over 24 h, while 2:1 reaction of cisplatin:rHA decreased the free thiol content of rHA by an average of 28% (Table II). Reaction of cisplatin with the two human serum albumin samples had no effect on the free thiol content. It is unlikely that the presence of protein-bound platinum influences the DTNB method, since the cisplatin adducts of SH-blocked rHA did not induce cleavage of DTNB.
This provides the first direct evidence that only a small fraction of cisplatin binds to Cys-34 in rHA and does not bind significantly to the SH group of HSA. In the abstract of their article, Momburg et al. (34) suggested the involvement of the SH group of HSA in cisplatin binding according to DTNB titration, but surprisingly, did not describe these results. Tosetti at al. (72) observed a 10-40% decrease of free SH content in the blood plasma of patients treated with cisplatin. In their work, the initial concentration of free thiol in blood plasma was about 300 µM, while the highest concentration of platinum in the samples did not exceed 4 µM. Therefore, the observed effects cannot be explained by direct interaction of cisplatin with protein thiols in blood plasma.Gel-filtration Chromatography-- It is known that platinum compounds can induce intermolecular cross-linking of proteins (73, 74). For example, albumin aggregation has been reported to arise from reaction of HSA with K2PtCl4 (75). FPLC data demonstrated that the reaction of cisplatin with rHA (1:1, 1 mM) was accompanied by a decrease in the amount of monomeric albumin to an average of 58%, by an increase of dimer content, and appearance of higher molecular mass polymers (Fig. 7B). These changes were also observed for 0.5:1 cisplatin:rHA reactions (data not shown) and were enhanced after reaction with a 2-fold molar excess of cisplatin (Fig. 7C). SH blocking of rHA reduced albumin aggregation, but did not prevent formation of significant amounts of dimers and polymers. These data suggest that cisplatin-induced albumin aggregation occurs mainly via direct intermolecular cross-linking, probably via monofunctional adducts or via NH3 release (species D in Scheme I).
It is noteworthy that we observed cisplatin-induced aggregation of albumin even at low cisplatin/albumin molar ratios. This observation may have important implications for the biological activity and therapeutic effects of cisplatin-albumin complexes such as those prepared at 1:1 cisplatin:albumin molar ratios by DeSimone et al. (29) and Holding at al. (30), or with a high molar excess of drug (7:1) by Hoshimo et al. (31). Cross-linked forms of albumin may not be effective delivery agents for cisplatin, being quickly eliminated from the circulation by hepatocytes and liver macrophages (76, 77). Cisplatin-induced protein cross-linking may also explain reported differences in the body distribution of platinum after infusion of free cisplatin or its complex with plasma proteins; in the former case, a major amount of platinum was detected in the kidney (78) and, in the latter, in liver tissue (20).Reaction of Cisplatin with Chemically Modified rHA in Media with Chloride
SH-blocked Albumin-- Carboxyamidomethylation of the free SH group with iodoacetamide (Fig. 8) or its modification with N-ethylmaleimide confirmed the assignment of cross-peaks a' and d' in the two-dimensional 1H,15N HSQC NMR spectrum for cisplatin bound to the SH group of rHA. SH blocking eliminated cross-peaks a' and d' but did not influence the chemical shifts and intensities of the other cross-peaks of protein-bound cisplatin (Fig. 8A). Additionally, modification of the free thiol decreased the rate of reaction of rHA with cisplatin, as was observed previously (33, 38). The decrease in the rate of this reaction may explain the decrease in the amount of dimer and polymer formed from reaction of SH-blocked rHA with cisplatin.
Methionine-modified rHA-- The involvement of methionine in cisplatin binding was demonstrated via methylation of methionines with iodomethane (forming -SMe2+ groups), which dramatically reduced the extent of cisplatin binding to albumin (Fig. 9A). Following modification, (i) only one pair of weak cross-peaks (e/f and c) was observed in the two-dimensional 1H,15N HSQC NMR spectrum after 13 h and (ii) most of the cisplatin was unbound over 17 h of reaction.
1H,15N NMR data for iodomethane-modified rHA strongly suggest that methionine is the sole source of sulfur in the major bifunctional (cross-peaks a and b) and two monofunctional (cross-peaks c, d, and e/f) cisplatin-albumin adducts. Weak signals e/f and c are likely to represent monofunctional cisplatin adducts formed by reaction with the small fraction of unmodified methionines or with disulfide groups.Histidine-modified Albumin--
Modification of six histidines of
rHA via reaction with diethylpyrocarbonate only slightly decreased the
intensities of 1H,15N cross-peaks c
and d and changed the 1H shift of cross-peak
e/f (Fig. 9B), assigned to a monofunctional adduct. It seems likely that the observed changes are due to indirect effects of histidine modification on cisplatin binding sites via small
changes in protein conformation. Since histidine modification induced a
dramatic decrease of the intensities of six relatively sharp signals in
the aromatic region of the 1H NMR spectrum of albumin
(CH), it is probable that the modification affects only
solvent-exposed His residues. Additionally, we did not observe any
significant changes to these peaks upon cisplatin binding; therefore,
imidazole rings of exposed histidines do not serve as nitrogen donors
in cisplatin adducts. X-ray crystallographic studies of several
proteins have suggested that only Met and His side chains exposed to
the solvent are able to interact with
[PtCl4]2
, while buried ones do not bind to
platinum (70). His residues of rHA which do not react with
diethylpyrocarbonate are probably buried inside the protein, and it
seems unlikely that these are involved in cisplatin binding, although
this cannot be ruled out. Backbone amide nitrogens or Lys or Arg side
chains with abnormally low pKa values may also serve
as possible N donors. Recently, it has been shown that a lysine amino
group in a high mobility group protein is involved in cisplatin binding
(79).
Possible Albumin Binding Sites for Cisplatin
The NMR results suggest that platinum reacts mainly with
methionine residues of rHA creating bifunctional (cross-peaks
a and b) and two monofunctional (peaks
c and d or c and e/f)
complexes as well as an adduct with Cys-34. Our data can be contrasted
with previous reports that a free thiol group in albumin is the major
site for cisplatin binding, based on experiments with blocking of
Cys-34 of HSA and BSA (7, 33, 38). However, albumin samples in the
previously reported experiments probably had a low initial level of
free thiol (can be as low as 0.3 mol mol1 protein; Table
II). It is also possible that bovine and human serum albumin have
different binding sites for cisplatin due to small differences in amino
acid sequence. In particular, HSA has six methionine residues, whereas
BSA has only four (substitutions M123L, L185M, M298I, M329S; Ref
21).
The crystal structure of human albumin has been published (21, 80); however, the atomic coordinates have not yet been made available. Attempts to locate possible platinum binding sites can therefore be made only on the basis of published ribbon diagrams of the structure of albumin, domain II and subdomains IIA and IIIB. These views were used to assess the positions and the relative solvent accessibilities of the six methionine side chains on human serum albumin, residues 87, 123, 298, 329, 446, and 548.
We can speculate that the major cisplatin binding site which appears to be an S,N macrochelate (peaks a and b) may utilize Met-298 as a ligand. Met-298 appears to be the most surface-accessible Met residue, and most likely accounts for the sharp singlet in the 1H NMR spectrum of albumin, which was suppressed upon cisplatin binding. The 1H chemical shift of the ammine trans to sulfur cross-peak for this macrochelate complex (4.50 ppm) is shifted significantly to higher frequency (~0.3 ppm) than has been observed for ammine trans to a methionine sulfur ligand (81). A possible reason for this is the presence of a H bond or salt-bridge interaction between the ammine hydrogen and nearby acceptor. Met-298 is in a region of high negative charge, with Asp-296 and Asp-301 nearby, together with glutamate residues 292, 294, and 297. Furthermore, this may be important for site preference if the reactant is positively charged, as is cis-[Pt(15NH3)2(H2O)2]2+ and monoaqua cisplatin.
Apart from Met-298, Met-87 and Met-446 may also be involved in the formation of monofunctional adducts with cisplatin since these appear to be exposed residues.
Conclusions
Contrary to previous reports, we find that the free thiolate group of Cys-34 of albumin is not the locus of the major cisplatin binding site. Our 1H,15N NMR data, obtained via the use of cis-[PtCl2(15NH3)2], combined with comparison of human serum albumin and recombinant human albumin, SH blocking, and His and Met modification reactions, suggest that the major binding site involves a Met S,N macrochelate, together with minor monofunctional sites involving Met S and Cys-34. The high trans influence of Cys S and Met S leads to the eventual displacement of platinum-bound NH3 ligands and protein cross-linking detectable by gel filtration chromatography. Platinum-induced formation of polymers may be of significance to the in vivo activity of cisplatin. The complexity of the reactions of cisplatin may explain why previous reports of the biological properties of cisplatin-albumin complexes have sometimes appeared to be contradictory.
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ACKNOWLEDGEMENTS |
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We thank the Medical Research Council Biomedical NMR Center (Mill Hill) and Engineering and Physical Sciences Research Council Ultrahigh Field NMR Center (Edinburgh) for the provision of NMR facilities.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council and Delta Biotechnology (CASE studentship to J.C.), Medical Research Council, and Royal Society (visiting fellowship to A. I. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Visiting scientist from the Institute of Bioorganic Chemistry, Belarus Academy of Sciences, Minsk, 220141 Belarus.
** To whom correspondence should be addressed: Dept. of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom. Tel.: 44-131-650-4729; Fax: 44-131-650-6452; E-mail: pjs01{at}holyrood.ed.ac.uk.
1 The abbreviations used are: HSA, human serum albumin; rHA, recombinant human albumin; BSA, bovine serum albumin; HSQC, heteronuclear single-quantum coherence; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); FPLC, fast protein liquid chromatography.
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REFERENCES |
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