This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
Acrylamide is widely used worldwide in industry and it can also be produced by the cooking and processing of foods. It is harmful to human beings, and human brain CK (HBCK) has been proposed to be one of the important targets of acrylamide. In this research, we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inactivated at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The binding sites of acrylamide were located at the cleft between the N- and C-terminal domains of CK, and Glu232 was one of the key binding residues. The effects of acrylamide on CK were proposed to be isoenzyme- and species-specific, and the underlying molecular mechanisms were discussed.
Acrylamide, an α,β-unsaturated reactive molecule [1], is widely used worldwide in industry. For example, it is used to synthesize polyacrylamide, a material that is frequently used in laboratories for gel electrophoresis experiments [2–4]. In daily life, acrylamide can be formed during the cooking and processing of foods via the decarboxylation of the Schiff base derived from carbonyl reactants, resulting in human consumption of relatively high doses of acrylamide [2–4]. The harmful effects of acrylamide have been proposed to be caused by its neurotoxicity [5–8], genotoxicity [9], carcinogenicity [10,11], reproductive and developmental toxicities [10] and cancer risk [11]. Among these toxic effects, epidemiological studies have shown that the neurotoxicity of acrylamide affects the nerve terminal functions. Acrylamide can modify the cysteine residues of presynaptic proteins, thereby significantly reducing the neurotransmitter release, which eventually leads to process degeneration [7,12]. Particularly, creatine kinase has been found to be significantly inhibited by acrylamide in the brain or sciatic nerve according to both in vitro and in vivo studies [13–15].
Creatine kinase (CK, EC 2.7.3.2), a member of the phosphagen kinase family, catalyzes the reversible phosphotransfer between the ATP/ADP and Cr/PCr systems [16]. CK is highly expressed in vertebrate excitable tissues that require large energy fluxes and plays a crucial role in intracellular energetics [20–22]. There are four major CK isoforms, which are named according to their tissue distribution or subcellular localization [17]. Two tissue-specific (muscle or brain) cytosolic CKs exist as homo- (MM and BB) or hetero-dimers (MB) composed of muscle (M) or brain (B) monomers, while two mitochondria isoenzymes can exist as dimer or octamer [18]. CKs share a high similarity in their primary sequences and core structures (Figure 1) [19,20], while the minor difference may correlate to their isoenzyme-specific functions [21]. The ubiquitous brain-type BB-CK is widely distributed in brain, heart, smooth muscle, nervous system and other tissues, whereas the muscle-type CK (MM-CK) is the predominant isoform in highly differentiated skeletal muscle tissue [18,22]. Due to its crucial role in vertebrate energy metabolism, BB-CK is involved in many vital physiological processes and serious diseases [22–29]. CK has also long been used as a model enzyme, and its catalytic mechanism, structure, stability and folding has been extensively studied [19].
Although BB-CK has been shown to be one of the targets of acrylamide [13,14], the underlying molecular mechanism is still unclear. Previous studies suggested that acrylamide can inhibit rabbit MM-CK (RMCK) and BB-CK (RBCK) by modifying the thiol groups of cysteines [15,30]. It is worth noting that BB-CK, but not MM-CK, is the predominant CK isoform in the brain and nervous system. Moreover, previous studies have identified the isoenzyme-specific functions and stability in CKs [21,31–33]. Thus it is worthwhile to investigate the effects of acrylamide on human BB-CK (HBCK) activity and structure. In this research, we investigated the effect of acrylamide on HBCK by activity assay, spectroscopic techniques and molecular simulation studies. The results herein are expected to further our understanding of the molecular mechanism of the toxic effects of acrylamide under physiological conditions.
Results and DiscussionInactivation of HBCK by Acrylamide
The effects of acrylamide on HBCK activity was evaluated by measuring the residual activity after incubation of the enzyme with various concentrations of acrylamide for 2 h. The data in Figure 2 clearly indicate that HBCK was inactivated by acrylamide in a dose-dependent manner. The catalytic activity of HBCK was completely eliminated by ∼300 mM acrylamide, and the IC50 value was ∼50 mM. It is worth noting that HBCK was much more easily to be inactivated by acrylamide than CKs from rabbit (RMCK and RBCK), which could still maintain about 10% of its activity at an acrylamide concentration of 600 mM and had an IC50 value of ∼200 mM [15]. Although it is not clear whether acrylamide is harmful to rabbit CKs, the results herein strongly suggested that the human BB-CK was much more sensitive to acrylamide-induced inactivation than CKs from rabbit.
The inactivation kinetics were measured by time-course inactivation studies in the presence of various concentrations of acrylamide, and the results are presented in Figure 3. The time-dependent decrease of the enzyme activity of HBCK was best-fitted to a biphasic process, and the fast- (k1) and slow-phase (k2) rate constants are presented in Table 1. The changes in the transition free-energy (ΔΔG°′) were also calculated by Equation 1 as described previously [30]:
ΔΔGo'=−RTlnk′where k′ is a time constant for the major phase (fast phase) of the inactivation reaction, T is the absolute temperature, and R is the Boltzmann constant. These results indicated that neither the inactivation rate constants nor ΔΔG°′ were significantly affected by the different concentrations of acrylamide, and the values were at the same order of magnitude.
Both RBCK and HBCK exhibited a biphasic process during acrylamide-induced inactivation, but they had different rate constants [30]. However, only a monophasic inactivation process was discovered for RMCK [15]. The difference might be caused by the relatively shorter time used for the study of RMCK or dissimilar inactivation mechanisms of the two isoenzymes by acrylamide. A comparison of the inactivation rate constants of HBCK/RBCK and RMCK indicated that although significant deviation was found for the fast phase rate constants, the slow phase rate constants of HBCK inactivation by acrylamide was almost identical for the three enzymes. It has been proposed that the inactivation of RBCK and RMCK by acrylamide was due to thiol depletion [15,30]. Because all CKs share a similar three-dimensional structure and the modification of thiol groups by acrylamide has the same rate constant, it is safe to conclude that the slow phase of HBCK inactivation was due to thiol depletion. This deduction was also supported by the fact that the rate constants of inactivation and thiol depletion were at the same level [15,30]. The fast phase inactivation of the two BB-CKs might be due to specific binding of the acrylamide molecules to the enzyme. The comparison of the inactivation processes of the three enzymes also suggested that the inactivation efficiency of acrylamide was isoenzyme- and species-dependent.
Effect of Acrylamide on the Tertiary Structure of HBCK
To elucidate the structural changes of HBCK by acrylamide, ANS extrinsic fluorescence was measured to probe the tertiary structural changes of HBCK induced by the addition of acrylamide. ANS has long been used as an extrinsic fluorescence probe to monitor the hydrophobic exposure of proteins [34]. As presented in Figure 4, the native HBCK exhibits typical ANS fluorescence emission maximum at around 470 nm, suggesting that, like MMCK [26,35–37], native BBCK molecules also contain ANS-binding sites.
In the presence of acrylamide, the intensity of ANS emission fluorescence increased slightly (∼20%) in an acrylamide concentration-dependent manner (inset of Figure 4), implying that the addition of acrylamide had a minor effect on the hydrophobic exposure of HBCK. At acrylamide concentrations above 256 mM, the ANS fluorescence had a significant blue-shift, accompanied by a decrease in intensity (data not shown), suggesting that high concentrations of acrylamide might interfere with the ANS fluorescence measurements.
It is worth noting that the tertiary structural changes of HBCK were different from those of RBCK, although they are highly homogenous in their primary sequences. Our previous study has shown that no significant change was observed at acrylamide concentrations below 800 mM for RBCK [30]. Their dissimilar structural stability coincided with their different response to acrylamide-induced inactivation.
Native-PAGE analysis was also performed to probe the change in the shape of the molecule (Figure 5), and the results were similar to those for RMCK and RBCK [15,30]. The shift of the band at acrylamide concentrations below 400 mM indicated that the tertiary structure of HBCK was modified by acrylamide, while the quaternary structure was not. The splitting of the band at acrylamide concentrations above 400 mM suggested that the thiol groups was alkylated by acrylamide, as proposed previously [30].
Acrylamide Binding-Site on HBCK by Docking Simulation
The above results from inactivation and structural studies suggested that acrylamide might have specific binding sites on HBCK. Actually, a previous study suggested that the acrylamide binding-site at RBCK was at a pocket around Cys74 according to docking simulation of a predicted RBCK structure [30]. Recently, the crystal structures of HBCK in ligand-free and ligand-binding forms have been published [20], and the results provided reliable structures for docking simulation studies. As presented in Figure 1, each subunit of CK contains two domains: a smaller N-terminal domain and a larger C-terminal domain, and the active site of CK is located at the cleft of the two domains [19]. This cleft or pocket is thought to facilitate the entry of substrates as well as inhibitors [19,30]. The HBCK pocket, which is estimated to be as large as ∼2,500 Å2, is shown in Figures 6A and 6B.
Three HBCK structures have been reported: the ligand-free (PDB ID: 3DRE), ADP-binding (PDB ID: 3DRB) and TSAC-binding (PDB ID: 3B6R) forms. To ensure the reliability of the docking results, all the crystal structures were used for the docking simulations, and no significant difference in the binding sites were found for the three crystal structures. The docking was also performed by both Autodock 4.0 and Fred 2.0 software programs, and the docking of the acrylamide molecule to the HBCK molecule was successful as indicated by the statistically significant scores (about −3.7 kcal/mol for Autodock and about −30 kcal/mol for Fred 2.0). Furthermore, we also tested the reliability by docking ADP to the ligand-free HBCK molecule, and the RMSD was 0.214 Å, which confirmed that the docking results were reliable.
Surprisingly, the two programs give different sites of acrylamide-binding (Figure 6). Considering that the weak binding of acrylamide and different CK isoforms had dissimilar IC50 values, it is possible that CK contains multiple acrylamide binding sites. Moreover, the biphasic inactivation process also suggested that BB-CK might have acryamide binding sites with distinct binding affinities. This could also explain why different sites were predicted by the two programs and why different sites were predicted for the two highly homologous BB-CK enzymes.
The binding site predicted by Autodock was formed by residues Pro143, Cys146, Arg151, Met207, Ala208, Arg209, Arg215, Asn230, Glu231, Glu232, Asp233 and His234, while that by Fred was formed by residues Thr133, Gly134, Arg135, Glu232, Asp233, Leu235, Arg236, Leu281, Asn286 and Gly 290. These two potential acrylamide binding sites were adjacent, and two residues (Glu232 and Asp233) were predicted to participate in acrylamide binding for both cases. Previous structural and mutational studies have pointed out that Glu232 is one of the crucial residues responsible for CK catalysis by participating in the positioning of creatine (Figure 1) [19,20,30]. Thus the binding of acrylamide might impair the structure and flexibility of the active site by altering the position of Glu232, resulting in HBCK inactivation.
A comparison of the results obtained herein and those of RMCK and RBCK [15,30] strongly suggested that the inhibition effect of acrylamide on CK might be isoenzyme- and species-specific. The structure and sequence are highly conserved among all CKs [19], and the identity in the primary sequence is 96.6% between HBCK and RBCK, and 80.1% between HBCK and RMCK. For all CKs, there is a large pocket between the N- and C-terminal domains to facilitate the entry of the substrates. Both the docking simulations in this research and that for RBCK suggested that the acrylamide binding sites were located at this pocket, but at different positions. These observations implied that acrylamide might have multi-binding sites on CK, while the affinity and position might influenced the structure of the active site in different ways. The disruption of the active site conformation might allow the modification of the thiol group of Cys283, a fully conserved residue crucial for CK, and further inactivate the enzyme. This proposal is consistent with the distinct IC50 values observed for the three CK isoenzymes, and could very well explain why different acrylamide binding sites were obtained for HBCK and RBCK by the two simulation programs. The species- and isoenzyme-specific response of CK against acrylamide-induced inactivation also suggested that great caution should be taken when using model proteins from other species to evaluate the effects of toxicants on human beings.
Experimental SectionMaterials and Protein Expression and Purification
Creatine, ATP, DTT, magnesium acetate, thymol blue, acrylamide, and ANS were all purchased from Sigma. All the other reagents were local products of analytical grade. Recombinant HBCK was expressed and purified as described previously [30,37]. HBCK purity was confirmed by the presence of only one band in both SDS- and native-PAGE analyses, as well as LC-MS/MS identification. The protein concentration was determined according to the Bradford method by using bovine serum albumin as a standard [38].
HBCK Activity Assay
HBCK activity was measured according to the pH-colorimetry method by following the proton generation during the reaction of ATP and creatine [39]. The enzymatic activity was monitored by the absorption at 597 nm with an Ultraspec 4300 pro UV/Visible spectrophotometer (Amersham Pharmacia Biotech), and the indicator was thymol blue. The reaction and measurements were performed at 25 °C. The assay buffer contained 24 mM creatine, 4 mM ATP, 8 mM magnesium acetate, 0.01% thymol blue, and 5 mM glycine-NaOH (pH 9.0).
ANS Fluorescence
The extrinsic fluorescence emission spectra were collected using a Hitachi F-2500 spectrofluorimeter (Tokyo, Japan) using 1-cm-pathlength cuvettes. The protein solutions with various amounts of acrylamide were incubated with 40 μM ANS for 30 min in the dark. An excitation wavelength of 390 nm was used for ANS-binding fluorescence, and the emission wavelength ranged from 400 to 600 nm.
In silico Docking of HBCK and Acrylamide
To ensure the reliability of the docking results, all of the three published HBCK crystal structures (PDB IDs: 3DRE, 3DRB and 3B6R) [20] were used for the in silico docking simulations. Two programs, Autodock 4.0 and Fred 2.0, which are based on different search techniques, were used in this research [40,41]. The original structure of acrylamide was obtained from the PubChem database (http://www.pubchem.org; compound ID: 6579) [42]. Details regarding the procedures of docking simulations were the same as those described previously [30,43]. The reliability of the simulations was also examined by docking the ADP molecule to the ligand-free HBCK molecule, and the RMSD was 0.214 Å, which confirmed that the docking results were reliable.
Conclusions
Acrylamide, which is widely used worldwide in industry and can be produced by the cooking and processing of foods, has been found to be harmful to human beings. BB-CK has been proposed to be one of the important targets in the neurotoxicity of acrylamide. In this research, we studied the effects of acrylamide on human BB-CK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inhibited at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The highly conserved structure of CKs but quite dissimilar responses suggested that the effects of acrylamide might be isoenzyme- and species-specific. The binding sites of acrylamide were proposed to be located at the cleft between the N- and C-terminal domains of CK, and the affinities of the multiple sites might be accounted for the slight difference in the sequence and structure of various CKs. This might be the structural basis of the different effect of acrylamide on various CKs. The results herein provide insight into the inhibitory effects and the possible ligand-binding mechanism of acrylamide on CK.
The authors thank Hai-Meng Zhou (Tsinghua University) for helpful discussions and comments about this work. This investigation was supported by the National Natural Science Foundation of China (No. 30500084), and the Science and Technology Planning Project of Jiaxing, Zhejiang, China (2008AZ1024).
ReferencesFriedmanMChemistry, biochemistry, and safety of acrylamide. A reviewJ. Agric. Food Chem2003514504452610.1021/jf030204+14705871RydbergPErikssonSTarekeEKarlssonPEhrenbergLTornqvistMFactors that influence the acrylamide content of heated foodsAdv. Exp. Med. Biol200556131732816438308TornqvistMAcrylamide in food: The discovery and its implications: A historical perspectiveAdv. Exp. Med. Biol200556111916438285BlankICurrent status of acrylamide research in food: Measurement, safety assessment, and formationAnn. N. Y. Acad. Sci20051043304010.1196/annals.1333.00416037219SmithEAOehmeFWAcrylamide and polyacrylamide: A review of production, use, environmental fate and neurotoxicityRev. Environ. Health199192152281668792LoPachinRMCanadyRAAcrylamide toxicities and food safety: Session IV summary and research needsNeurotoxicology20042550750910.1016/j.neuro.2003.12.00215264346LoPachinRMThe changing view of acrylamide neurotoxicityNeurotoxicology20042561763010.1016/j.neuro.2004.01.00415183015LoPachinRMSchwarczAIGaughanCLMansukhaniSDasSIn vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and releaseNeurotoxicology20042534936310.1016/S0161-813X(03)00149-915019298BesaratiniaAPfeiferGPDNA adduction and mutagenic properties of acrylamideMutat. Res2005580314010.1016/j.mrgentox.2004.10.01115668105DearfieldKLAbernathyCOOttleyMSBrantnerJHHayesPFAcrylamide: Its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicityMutat. Res1988195457710.1016/0165-1110(88)90015-23275881RudenCAcrylamide and cancer risk—expert risk assessments and the public debateFood Chem. Toxicol20044233534910.1016/j.fct.2003.10.01714871575LoPachinRMBarberDSSynaptic cysteine sulfhydryl groups as targets of electrophilic neurotoxicantsToxicol. Sci20069424025510.1093/toxsci/kfl06616880199MatsuokaMIgisuHLinJInoueNEffects of acrylamide and N,N’-methylene-bis-acrylamide on creatine kinase activityBrain Res199050735135310.1016/0006-8993(90)90297-O2337777KohriyamaKMatsuokaMIgisuHEffects of acrylamide and acrylic acid on creatine kinase activity in the rat brainArch. Toxicol19946867708166608MengFGZhouHWZhouHMEffects of acrylamide on creatine kinase from rabbit muscleInt. J. Biochem. Cell Biol2001331064107010.1016/S1357-2725(01)00079-611551822WattsDCCreatine Kinase (Adenosine 5′-Triphosphate Creatine Phosphotransferase)Academic PressNew York, NY, USA1973383455WallimannTWyssMBrdiczkaDNicolayKEppenbergerHMIntracellular compartmentation, structure and function of creatine kinase isozymes in tissues with high and fluctuating energy demands: The “phosphocreatine circuit” for cellular energy homeostasisBiochem. J199228121401731757EppenbergerHMDawsonDMKaplanNOThe comparative enzymology of creatine kinases. I. Isolation and characterization from chicken and rabbit tissuesJ. Biol. Chem1976108377392McLeishMJKenyonGLRelating structure to mechanism in creatine kinaseCrit. Rev. Biochem. Mol. Biol20054012010.1080/1040923059091857715804623BongSMMoonJHNamKHLeeKSChiYMHwangKYStructural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3-creatine transition-state analogue complexFEBS Lett20085823959396510.1016/j.febslet.2008.10.03918977227HornemannTStolzMWallimannTIsoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH2-terminal lysine charge-clampsJ. Cell Biol20001491225123410.1083/jcb.149.6.122510851020WallimannTHemmerWCreatine-kinase in nonmuscle tissues and cellsMol. Cell Biochem199413319322010.1007/BF012679557808454AksenovMAksenovaMButterfieldDAMarkesberyWROxidative modification of creatine kinase BB in Alzheimer’s disease brainJ. Neurochem2000742520252710820214ButterfieldDAKanskiJBrain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteinsMech. Ageing Dev200112294596210.1016/S0047-6374(01)00249-411348660GallantMRakMSzeghalmiADel BigioMRWestawayDYangJJulianRGoughKMFocally elevated creatine detected in amyloid precursor protein (APP) transgenic mice and Alzheimer disease brain tissueJ. Biol. Chem20062815816267054FengSZhaoT-JZhouH-MYanY-BEffects of the single point genetic mutation D54G on muscle creatine kinase activity, structure and stabilityInt. J. Biochem. Cell Biol20073939240110.1016/j.biocel.2006.09.00417030001ShinJ-BStreijgerFBeynonAPetersTGadzalaLMcMillenDBystromCvan der ZeeCEEMWallimannTGillespiePGHair bundles are specialized for ATP delivery via creatine kinaseNeuron20075337138610.1016/j.neuron.2006.12.02117270734AndresRHDucrayADSchlattnerUWallimannTWidmerHRFunctions and effects of creatine in the central nervous systemBrain Res. Bull20087632934310.1016/j.brainresbull.2008.02.03518502307Salin-CantegrelAShekarabiMHolbertSDionPRochefortDLaganiereJDacalSHincePKaremeraLGasparCLapointeJ-YRouleauGAHMSN/ACC truncation mutations disrupt brain-type creatine kinase-dependent activation of K+/Cl− co-transporter 3Hum. Mol. Genet2008172703271110.1093/hmg/ddn17218566107LuZ-RZouH-CParkSJParkDShiLOhSHParkY-DBhakJZouFThe effects of acrylamide on brain creatine kinase: Inhibition kinetics and computational docking simulationInt. J. Biol. Macromol20094412813210.1016/j.ijbiomac.2008.11.00319061912SchlattnerUReinhartCHornemannTTokarska-SchlattnerMWallimannTIsoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display libraryBiochim. Biophys. Acta2002157912413210.1016/S0167-4781(02)00530-412427547WendtSSchlattnerUWallimannTDifferential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes—Inactivation, octamer destabilization, and identification of involved residuesJ. Biol. Chem20032781125113010.1074/jbc.M20857220012401781GuoZWangZWangXCStudies on the stability of creatine kinase isozymesBiochem. Cell Biol20038191610.1139/o02-17112683631RosenCWeberGDimer formation from 1-amino-8-naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding propertiesBiochemistry196983915392010.1021/bi00838a0065388144HeH-WFengSPangMZhouH-MYanY-BRole of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinaseInt. J. Biochem. Cell Biol2007391816182710.1016/j.biocel.2007.04.02817616428LiuY-MFengSZhaoT-JDingX-LYanY-BThe conserved Cys254 plays a crucial role in creatine kinase refolding under non-reduced conditions but not in its activity or stabilityBiochim. Biophys. Acta - Proteins Proteomics200817842071207810.1016/j.bbapap.2008.08.018ZhaoT-JFengSWangY-LLiuYLuoX-CZhouH-MYanY-BImpact of intra-subunit domain-domain interactions on creatine kinase activity and stabilityFEBS Lett20065803835384010.1016/j.febslet.2006.05.07616797013BradfordMMA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnal. Biochem19767224825410.1016/0003-2697(76)90527-3942051YaoQ-ZZhouHHouLTsouC-LA comparison of denaturation and inactivation rates of creatine kinase in guanidine solutionsSci. Sin198225B12961302McGannMRAlmondHRNichollsAGrantJABrownFKGaussian docking functionsBiopolymers200368769010.1002/bip.1020712579581HueyRMorrisGMOlsonAJGoodsellDSA semiempirical free energy force field with charge-based desolvationJ. Comput. Chem2007281145115210.1002/jcc.2063417274016XieXQChenJZData mining a small molecule drug screening representative subset from NIH PubChemJ. Chem. Inf. Model20084846547510.1021/ci700193u18302356HendlichMRippmannFBarnickelGLIGSITE: Automatic and efficient detection of potential small molecule-binding sites in proteinsJ Mol Graph Model199715359363389Figures and Table
Structure of HBCK monomer (PDB ID: 3B6R) [20]. N and C denote the N- and C-terminus of the protein. The positions of the substrates are highlighted by the stick model.
Effect of acrylamide on the activity of HBCK. The residual activity was measured after 2 h incubation of HBCK in 50 mM Tris-HCl buffer, pH 8.0, with the addition of various concentrations of acrylamide at 25 °C. The final enzyme concentration was 2 μM. The data are presented as average ± standard errors for three repetitions.
Inactivation kinetics of HBCK by various concentrations of acrylamide ranging from 0 to 800 mM. The enzyme solutions were mixed with various concentrations of acrylamide, and aliquots were taken at the indicated time points. Then the residual activity was measured using the standard activity assay, and the data were normalized by taking the activity recorded at 0 min as 100%. The data were fitted by a biphasic process, and the fitted data are presented as solid lines. The rate constants are presented in Table 1.
Effect of acrylamide on the ANS fluorescence of HBCK. The enzyme solutions were mixed with various concentrations of acrylamide and equilibrated for 2 h. The final concentration of ANS was 40 μM, and the solutions were incubated at ambient temperature for 30 min in the dark before measurements. The final enzyme concentration was 2 μM. The presented spectra were obtained by subtracting the spectra of ANS in the same buffer.
Native-PAGE analysis of the tertiary structural changes of HBCK induced by acrylamide. The protein was dissolved in 50 mM Tris-HCl buffer, pH 8.0, in the presence of various concentrations of acrylamide. Lanes 1–6 indicate the protein incubated in the buffer with the addition of 0, 50, 100, 200, 400 and 800 mM acrylamide, respectively.
The surface of HBCK (A and B) and acrylamide binding sites predicted by Autodock (C and D) and Fred (E and F). (A and B) The surface of the HBCK molecule. The yellow parts indicated the position of the cleft or pocket between the two domains of HBCK. Panel (B) shows the top view of the structure shown in panel (A). (C-F) The residues forming the binding site are shown by a line model, the acrylamide molecule is presented by a space-filling model, while Glu232 is highlighted by a stick model.
Inactivation rate constants of HBCK in the presence of acrylamide.