Concentrations Of Guanidine Hydrochloride Research Articles

Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy

In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropically induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the presence of an intermediate state during denaturation. A global analysis using non-linear regression gave a DeltaG(H(2)O,D)(0) = 6.7 kcal x mol(-1) for the complete unfolding of the BSA-ANS complexes, and a DeltaG(H(2)O,I) = 0.9 kcal x mol(-1) for the first step to the intermediate. Therefore, the unfolding energy of the intermediate, which appears mostly at intermediate GuHCl concentrations (1.0 to 1.5 M), to the denatured state, is 5.8 kcal x mol(-1). The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (<1.5 M).

Structural Insights into Alternate Aggregated Prion Protein Forms

The conversion of the cellular form of the prion protein (PrP C) to an abnormal, alternatively folded isoform (PrP Sc) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies. In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.

Biophysical Characterization and Formulation of F1–V, a Recombinant Plague Antigen

The recombinant plague antigen, F1–V, was studied for its structural characteristics using several biophysical techniques. A larger apparent molecular weight relative to its calculated molecular weight obtained from size exclusion chromatography, an unusually large Rg obtained from MALS, and ANS dye binding studies which indicate that all hydrophobic regions of the protein are exposed to solvent demonstrated that F1–V exists like a disordered protein with a worm-like conformation. The pH-solubility profile of F1–V showed a solubility minimum at pH 5, close to its pI, consistent with the lack of repulsive forces that result in aggregation. Thus, in contrast to most globular proteins that exhibit a secondary and a tertiary structure, F1–V seems to lack tertiary structure and like an unfolded protein is more prone to aggregation via hydrophobic interactions. Despite this, when renatured gradually using descending guanidine hydrochloride concentration dialysis, in the presence of Mg+2, a surfactant and arginine hydrochloride at a pH of 7.5, F1–V appears to populate predominantly in its monomeric state. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2592–2602, 2009

Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243-249, 310-312, and 386-400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.

Improved Solubilization of Recombinant Human Growth Hormone Inclusion Body Produced inEscherichia coli

Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGH-dependent cell line.

Unfolding and Inactivation of Abalone (Haliotis diversicolor) Alkaline Phosphatase During Denaturation by Guanidine Hydrochloride

Abalone, a kind of low poikilothermic invertebrate, is easily exposed to ocean environment stress. Since it is one of the important mariculture animals, the attention paid to the abalone study becomes increasing. Alkaline phosphatase (ALPase, EC 3.1.3.1) is a kind of zinc-contained metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. Unfolding and inactivation of ALPase from abalone (Haliotis diversicolor) during denaturation by guanidine hydrochloride (GuHCl) of different concentrations has first been studied. The kinetic theory of the substrate reaction by enzyme was described by Tsou, which was applied to the study on ALPase's kinetic course of inactivation by GuHCl. The result showed that the inactivation of the enzyme by GuHCl was a slow, reversible reaction with fractional remaining activity. The microscopic rate constants were determined. The result, k(+0) > k'(+0), showed that the enzyme was protected by the substrate to a certain extent during guanidine denaturation. The changes of conformation of the enzyme in different concentrations of GuHCl have been studied by means of measuring the fluorescence spectra. The results showed that the inactivation occurred before the noticeable conformational changes of the enzyme molecule as a whole can be detected, which suggests that the active site of the enzyme has more flexibility than the whole enzyme molecule. These studies will facilitate the understanding of physiological and biochemical features of the H. diversicolor and will also help in the understanding of the abalone immune system.

Refolding of proteins from inclusion bodies is favored by a diminished hydrophobic effect at elevated pressures

The application of high hydrostatic pressure is an effective tool to promote dissolution and refolding of protein from aggregates and inclusion bodies while minimizing reaggregation. In this study we explored the mechanism of high-pressure protein refolding by quantitatively assessing the magnitude of the protein-protein interactions both at atmospheric and elevated pressures for T4 lysozyme, in solutions containing various amounts of guanidinium hydrochloride. At atmospheric pressure, the protein- protein interactions are most attractive at moderate guanidinium hydrochloride concentrations (approximately 1-2 molar), as indicated by a minimum in B(22) values. In contrast, at a pressure of 1,000 bar no minimum in B(22) values is observed, indicating that high pressures colloidally stabilize protein against aggregation. Finally, experimental values of refractive index increments as a function of pressure indicate that at high pressures, wetting of the hydrophobic surfaces is favored, resulting in a reduction of the hydrophobic effect. This reduction in the hydrophobic effect reduces the driving force for aggregation of (partially) unfolded protein.

Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride

To find out the changes in the internal dynamics and function of proteins as a consequence of their binding interactions with guanidine hydrochloride (GdnHCl), laser flash photolysis and optical absorption methods have been used to study the dynamic events in the horse myoglobin-CO complex (MbCO) in the presence of subdenaturing concentrations of GdnHCl at pH 7, 22 degrees C. The rate coefficients for geminate rebinding and bimolecular rebinding (k(on)) were measured by laser photolysis of CO in MbCO, and the CO dissociation rate (k(off)) was determined by the CO replacement method using hexacyanoferrate ion or NO. Starting from the native-state condition, the values of k(on) and k(off) decrease by approximately 1.4 (+/-0.1)-fold in the presence of 0.1-0.3 M GdnHCl, and then increase at higher concentrations of the denaturant. This has been taken as evidence for internal motional constraints and increased stability of the protein in the subdenaturing region giving rise to gated entry of the photolyzed CO from the solvent. The rate for geminate rebinding does not show any decrease in the rate versus GdnHCl concentration plots. The values for the activation enthalpy for the CO dissociation reaction and the entropy loss relative to the native-state entropy, both measured as a function of GdnHCl concentration, indicate that the protein is indeed stabilized under subdenaturing conditions. Analyses of thermal unfolding transitions of myoglobin in the presence of different concentrations of GdnHCl indicate that the stability of this protein extracted from the linear free energy model is approximately 3-4 kcal mol(-1) less than the true stability. The results indicate the appropriateness of the denaturant binding model for the analysis of GdnHCl-induced unfolding data, and provide a value of 7.9 kcal mol(-1) as the true stability of the protein.

“Iron priming” guides folding of denatured aporubredoxins

The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys)(4) sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys)(4) sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys)(4) site must attain the ferric state in order to achieve its native structure.

Refolding of endostatin from inclusion bodies using high hydrostatic pressure

High hydrostatic pressure was used for concomitant solubilization and refolding of insoluble endostatin (ES) aggregated as inclusion bodies (IBs). High hydrostatic pressure (200 MPa or 2 kbar) was applied in combination with nondenaturing concentrations of guanidine hydrochloride. High levels of correctly folded ES (90 mg/L culture) were obtained after optimization/standardization of the procedure by applying pressures of 200 MPa for 16 h in 1.5 M guanidine hydrochloride/0.5 mM oxidized glutathione and reduced glutathione. Refolded ES was purified by affinity chromatography on a heparin column and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting, size exclusion HPLC, circular dichroism, and intrinsic fluorescence. We demonstrated that high pressure can successfully convert insoluble IBs of ES expressed in Escherichia coli into an ES preparation with native tertiary structure and full biological activity.

Mechanism for stabilization of the molten globule state of papain by sodium n-alkyl sulfates: Spectroscopic and calorimetric approaches

Papain exists in a molten globule (MG) state at pH 2 and in this state protein tends to aggregate in the presence of lower concentrations of guanidine hydrochloride (GuHCl). Such aggregation is prevented if low concentrations of sodium n-alkyl sulfates are also present in the buffer; in addition, stabilization of the protein is also induced. The guanidine hydrochloride and temperature-induced unfolding of papain, in the presence of n-alkyl sulfates, indicate stabilization of the protein as seen from the higher transition midpoints when monitored by fluorescence, circular dichroism, and differential scanning calorimetry. However, a similar phenomenon is not seen under neutral conditions in the presence of n-alkyl sulfate concentrations. The effect of n-alkyl sulfates on the structure of the MG state of papain was utilized to investigate the contribution of hydrophobic interaction to the stability of the MG state. The T d values of the MG states of papain in the presence of n-alkyl sulfates at different concentrations showed substantial variation. The enhancement of T d values at the stability criterion of MG states corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the hydrophobic interactions play important roles in stabilizing and preventing the aggregation of the MG state of papain.

Submolecular unfolding units of Pseudomonas aeruginosa cytochrome c-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function.

Molecular cloning and characterization of Plasmodium falciparum transketolase

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K m app values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K i value of 305 μM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70 kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% α-helices and 26% β-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.

Denaturant-induced Expansion and Compaction of a Multi-domain Protein: IgG

It is generally believed that unfolded or denatured proteins show random-coil statistics and hence their radius of gyration simply scales with solvent quality (or concentration of denaturant). Indeed, nearly all proteins studied thus far have been shown to undergo a gradual and continuous expansion with increasing concentration of denaturant. Here, we use fluorescence correlation spectroscopy (FCS) to show that while protein A, a multi-domain and predominantly helical protein, expands gradually and continuously with increasing concentration of guanidine hydrochloride (GdnHCl), the F(ab′)2 fragment of goat anti-rabbit antibody IgG, a multi-subunit all β-sheet protein does not show such continuous expansion behavior. Instead, it first expands and then contracts with increasing concentration of GdnHCl. Even more striking is the fact that the hydrodynamic radius of the most expanded F(ab′)2 ensemble, observed at 3–4 M GdnHCl, is ∼3.6 times that of the native protein. Further FCS measurements involving urea and NaCl show that the unusually expanded F(ab′)2 conformations might be due to electrostatic repulsions. Taken together, these results suggest that specific interactions need to be considered while assessing the conformational and statistical properties of unfolded proteins, particularly under conditions of low solvent quality.

Identification of three phases in Onconase refolding

Onconase is an extremely stable member of the RNase A superfamily. The increase in the thermodynamic stability by 20 kJ x mol(-1) in comparison to RNase A was expected to result in altered folding behavior. Despite the lack of cis-Pro residues in native Onconase, refolding at low concentrations of guanidine hydrochloride was complex and showed three kinetic phases (fast, medium, and slow), with rate constants differing by a factor of about 10 each. None of the phases could be accelerated by peptidyl-prolyl cis-trans isomerases, pointing to the absence of kinetic phases that are limited by Pro isomerization. The detailed analysis by various probes indicates that the burial of the N-terminal Trp3, which is associated with the restoration of the active site, occurs in the slow phase, i.e. in the last step of refolding. Evidently, in contrast to the folding of RNase A, there is no catalytically active native-like intermediate in the folding of Onconase.

Study of thermally and chemically unfolded conformations of bovine serum albumin by means of dynamic light scattering

The change of conformation of a bovine serum albumin (BSA) has been characterized using dynamic light scattering (DLS). Structural properties were investigated as a function of chemical denaturant concentrations and a temperature. In pure water, the protein keeps its native size at guanidine hydrochloride (GdmCl) concentrations below 1 M and behaves like an excluded volume chain above 4 M. A transition in structure and properties of the protein seems to occur around a GdmCl concentration of 1.7 M. Still, this protein forms an unfolded conformation in presence of 6 M of urea concentration and the value of the diffusive virial coefficient indicates that the interactions between the polypeptide chain and solvent are repulsive. The true value of the T m = 43 °C, has obtained by linear extrapolation to 0 M GdmCl.

Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants

Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yañez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956-6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1-C2 or the C1-C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size-exclusion chromatographic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum approximately 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose-1,6-bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a 'phase diagram' reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1-C2 interface.

Impact of cofactor on stability of bacterial (CopZ) and human (Atox1) copper chaperones

Here, we present the first characterization of in vitro unfolding and thermodynamic stability of two copper chaperone proteins: Bacillus subtilis CopZ and Homo sapiens Atox1 . We find that the unfolding reactions for apo- and Cu(I)-forms of CopZ and Atox1, induced by the chemical denaturant, guanidine hydrochloride (GuHCl), and by thermal perturbation are reversible two-state reactions. For both proteins, the unfolding midpoints shift to higher GuHCl concentrations and the thermodynamic stability is increased in the presence of Cu(I). Despite the same overall fold, apo-CopZ exhibits much lower thermal stability than apo-Atox1. Although the thermal stability of both proteins is increased in the presence of copper, the stabilizing effect is largest for the less stable variant. Divergent energetic properties of the apo- and holo-forms may be linked to conformational changes that facilitate copper transfer to the target.

Kinetic stabilities of soybean and horseradish peroxidases

Peroxidases have attractive biocatalytic properties and are used in biosensing and immunoassays. Among various peroxidases, isoenzyme C of horseradish peroxidase (HRP-C) is the most studied, and is also the most commercially used due to its high structural stability. Soybean peroxidase (SBP) and horseradish peroxidase share strikingly similar three-dimensional structures with ∼60% sequence homology. We reported previously, that the conformational and thermal stabilities of SBP are substantially higher than HRP-C. In the present study, we show that the kinetic stability of SBP is much higher than HRP-C as obtained by measuring their unfolding rates at various guanidine hydrochloride (GdnHCl) concentrations. In contrast, the heme-free forms of SBP and HRP-C showed similar kinetic stabilities. We conclude that the higher structural stability of SBP compared to HRP-C stems from the heme binding to the apo protein. Commercial interest of these results is twofold. A cheaper, abundant, better active, and more stable SBP could replace HRP-C. The stability and hence the biocatalytic property of a peroxidase can be improved by suitably engineering the heme active-site that enhances the heme-apo-protein interaction.

Inhibitor‐mediated stabilization of the conformational structure of a histone deacetylase‐like amidohydrolase

Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.