Naoko Ohta

Effect of trans-diamminedichloroplatinum(II) on human serum albumin : conformational changes through partial disulfide bond cleavage

Abstract trans -Diamminedichloroplatinum(II) ( trans -DDP), the trans isomer of cis -diamminedichloroplatinum(II) ( cis -DDP), was bound to human serum albumin (HSA) in pH 7.4 buffer containing 0.1 M NaCl at 37°C. The amount of bound trans -DDP per mol of HSA was found to be 21.4 mol when the protein was incubated with a 40-fold excess of trans -DDP for 6 days. In trans -DDP-treated HSA, 3.4 disulfide (S-S) bonds were cleaved, where one HSA molecule contains 17 S-S bonds. The spectral characteristics of trans -DDP-treated HSA were examined in terms of the fluorescence spectrum of its lone tryptophan (Trp-214), and molar ellipticity. The relative fluorescence intensity of platinum-bound HSA decreased to 32.4% of that of the native state, suggesting that perturbation around the Trp-214 residue took place. This was confirmed by the destruction of the warfarin-binding site containing Trp-214 observed in the metal-bound HSA. Analysis of circular dichroism (CD) spectra showed a decreasing helix content from 50.5% in the native state to 30.6% in the metal-bound HSA. These conformational changes observed in HSA may be attributed to the S-S bond rupture induced by trans -DDP. Comparison with cis -DDP, which has already been shown to cleave S-S bonds in HSA, revealed that trans -DDP binds to HSA and cleaves S-S bonds more readily than the cis isomer.

Disulfide bond cleavage of human serum albumin and alterations of its secondary structure by cis-diamminedichloroplatinum(II)

Abstract cw -Diamminedichloroplatinum(II) ( cis -DDP) cleaved disulfide (S-S) bonds in human serum albumin (HSA) and brought about alterations of the secondary structure. The α-helix content decreased from 50.5% (native) to approx. 33% (four S-S bonds cleaved). The tendency toward a decrease corresponded only with an increase in the β-sheet. Sulfitolysis of the S-S bonds showed a tendency similar to that of metal binding. Fluorescence and UV difference spectra changed as a function of S-S bond cleavage and led to considerable differences between the two cleaving agents.

Reaction of oxidized glutathione with cis- and trans-diamminedichloroplatinum(II) and their aquated complexes through the disulfide bond

Abstract The conjugation of cis-diamminedichloroplatinum(II) (cis-DDP) by reduced glutathione (GSH) has been reported in several studies. In this study, oxidized glutathione (GSSG) was found to interact with platinum complexes through the S–S bond. The following platinum complexes were used: cis-DDP, trans-DDP and their aquated complexes. The second-order rate constants for the reactions of cis-DDP and its aquated complexes with S–S in GSSG at pH 7.4 and 37°C were 4.12×10−3 and 3.56×10−2 M−1 s−1, respectively. The rate constants for the reactions of trans-DDP and its aquated complexes with GSSG were 1.59×10−2 and 1.24×10−1 M−1 s−1, respectively. In order to compare the reactivity of GSH with GSSG, the rate constant for the decrease in GSH sulfhydryl by cis-DDP and its aquated complexes were obtained as 7.11×10−2 and 2.13×10−1 M−1 s−1. The rate constant for the reaction of GSH toward cis-DDP was 17-fold larger than that of GSSG, while a 6-fold larger rate constant was obtained for the aquated complexes of cis-DDP. cis-[Pt(NH3)2Cl(H2O)]+, the main species at pH 4.5, showed a 2-fold larger rate constant than cis-[Pt(NH3)2Cl(OH)] at pH 9.5. The difference between the two pHs may reflect that water is a better leaving group than the hydroxide ligand. These results suggest that GSSG may be involved in the conjugation of cis-DDP like GSH.

Degradation of gabexate mesilate catalyzed by human serum albumin

Abstract The degradation of gabexate mesilate (GM) was accelerated in the presence of human serum albumin (HSA), and was investigated based on Michaelis-Menten-type kinetics involving the formation of HSA-drug complex. The dissociation constant of the complex was 1.19 × 10 −4 M and the degradation rate constant of the complex was 1530-fold larger than the spontaneous rate constant (2.23 × 10 −6 s −1 ) at pH 7.4 (25°C). Potential active sites appeared to be histidine residues since the pH profiles indicated the involvement of a group with a pK a of about 6 and HSA modified with diethylpyrocarbonate (DEP) showed diminished activity. From the relationship between the number of histidine residues modified by DEP and the activity remaining in the partially modified HSA, only one histidine residue was involved in the degradation of GM. The lack of deuterium solvent isotope effect and the accumulation of acylated-HSA intermediate indicated that the enhanced rate of degradation is due to nucleophilic catalysis perhaps by the imidazole group. The degradation rate of GM was faster in human plasma than in HSA where the albumin concentration was adjusted to that in the plasma. The contribution of HSA mediated degradation to the overall degradation in human plasma was estimated about 40%. The results suggest that further attention should be paid to the non-specific esterase-like activity of HSA toward ester drugs especially with a good leaving group as far as drug disposition in plasma is concerned.

Aggregation of γ-globulin by cis-diamminedichloroplatinum(II): Alteration of Fc region and restoration by diethyldithiocarbamate

Abstract Human γ-globulin containing IgGs generally shows a binding ability to protein A through its Fc region. When purified human γ-globulin was incubated with cis -diamminedichloroplatinum(II) ( cis -DDP), reduced binding to protein A was observed. On the other hand, γ-globulin in human plasma showed only a slight decrease in protein A binding at similar doses, due probably to other internal substances in plasma by trapping cis -DDP. Reduction of γ-globulin pretreated with cis -DDP resulted in significantly decreased amounts of the H and HL components, whereas the L chain was normally detected. These results suggest that cis -DDP affects the disulfide(S-S) bond(s) in the inter H-H chains which locate in the Fc region. We have demonstrated that cis -DDP causes y-globulin polymerization and its S-S bond cleavage (Chen et al., Int. J. Pharm., 106 (1994) 249–253). Diethyldithiocarbamate (DDTC) partly restored these effects of cis -DDP on γ-globulin in terms of the decreased S-S bonds, polymerization, and the reduced binding ability of y-globulin to protein A. Since DDTC is known to easily cleave the Pt-S bond, Pt-S bonds are likely to be responsible for the restoration of this γ-globulin- cis -DDP interaction.