superoxide dismutases ( sod , ec 1.15.1.1 ) are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide [ 13 ] . they play an important antioxidant defense role in skins exposed to oxygen . in this regard , for the treatment of systemic inflammatory diseases including skin ulcer lesions , the topical application of free mn - sod or cu , zn - sod extracted from bovine , bacterial , and other species was dramatically effective in skin lesions . it has been reported that significant increase in the levels of sod occurs in vitiligo patients due to the increased oxidative stress . the involvement of oxidative stress in chronic idiopathic urticaria associated with sod was also reported : the activity of sod was markedly increased in lesional skin as compared with skin of healthy subjects , indicating that oxidative stress is crucially involved in chronic idiopathic urticaria and suggesting that oxidative stress is secondary to the development of inflammation . the earlier reports [ 7 , 8 ] suggested that the activity of activator protein-1 , which is associated with tumor promotion , was reduced in mn - sod transgenic mice overexpressing mn - sod in the skin , suggesting that mn - sod reduced tumor incidence by suppressing activator protein-1 activation . the mechanism of mn - sod catalysis is very important , and the mechanism therefore needs to be investigated from different sources using various kinetic methods . the information regarding the tertiary structure and the structural integrity of the active site of mn - sod is little known and in this regard , investigation on structure - function relationships in this enzyme including docking of a ligand is important . in this study , we applied al to understand mn - sod structural changes and inhibition mechanisms . as a result , we proposed an inhibitory effect of al on mn - sod and suggest the mechanisms of combination between inhibition kinetics and computational prediction to depict the al action in the catalysis of mn - sod . aluminum chloride crystal ( alcl36h20 ) , pyrogallol , and ans were purchased from sigma - aldrich ( usa ) . the crude form of mn - sod ( from thermus thermophilus ) was purchased from biotech company ( china ) . we further purified mn - sod using the ktafplc system ( ge healthcare , usa ) ; a single band was obtained on both sds - page and native nonreducing page gels . 10  mm tris - hcl buffer ( ph 8.2 ) was used during preparing all samples in this study . the assay for mn - sod the activity of sod was calculated according to the procedures of pyrogallol 's autoxidation , which could be monitored by the change in absorbance at 325  nm per min . reactions were performed in a typical reaction volume of 1 ml to which 10  l of enzyme solution was added to measure mn - sod activity . the activity and absorption were measured with a perkin elmer lambda bio u / v spectrophotometer . far - uv cd spectra of mn - sod at different al concentrations were recorded on a jasco j-810 spectropolarimeter in the region of 190250  nm at room temperature . mn - sod was denatured by incubation in 10  mm tris - hcl ( ph 8.2 ) containing various concentrations of al for 3  h , 25c . the fluorescence emission spectra were measured with a jasco fp750 spectrofluorometer with the use of a 1  cm path - length cuvette . an excitation wavelength of 280  nm was used for the tryptophan fluorescence measurements , and the emission wavelength ranged between 300 and 410  nm . the changes of the ans - binding fluorescence intensity for mn - sod were studied by labeling with 40  m ans for 30 min prior to measurement . an excitation wavelength of 380  nm was used for the ans - binding fluorescence , and the emission wavelength ranged from 400 to 650  nm . according to a previous report , when small molecules are bound to equivalent sites on a macromolecule , the equilibrium between free and bound molecules are given by the following equation to evaluate the binding constant ( k ) and number of binding sites ( n ) : 
								 ( 1)f0f0f=1n+1k1[q ] , 
							 where f0 and f are the relative steady - state fluorescence intensities in the absence and presence of quencher , respectively . the values for k and n can be derived from the intercept and slope of a plot based on ( 1 ) . the known 3d structure of mn - sod was obtained from pdb data base ( i d : 3mds ) . among the many tools available for in silico protein - ligand docking , the program performed ligand docking using a set of predefined 3d grids of the target protein and used a systemic search technique . the original structure of al was derived from the pubchem database ( compound i d : 104727 , http://www.pubchem.org/ ) . to prepare for the docking procedure , the following steps were taken : ( 1 ) conversion of 2d structures to 3d structures , ( 2 ) calculation of charges , ( 3 ) addition of hydrogen atoms , and ( 4 ) location of pockets . for these steps , we used the fconverter program of the j - chem package ( http://www.chemaxon.com/ ) and openbabel ( http://openbabel.org/ ) . we assayed mn - sod at the equilibrium and the kinetic states in the presence of al . mn - sod was significantly inactivated by al with a dose - dependent manner ( figure 1 ) . when the al concentration was increased to 0.8  mm the ic50 value was measured as 0.19  mm ( n = 2 ) .     to evaluate the the different time courses of mn - sod in the presence of 0.2 , 0.3 , and 0.4  mm al , respectively , were recorded ( figure 2(a ) ) . the enzyme activity was gradually decreased with time intervals and this implied that al may induce mn - sod tertiary structural change due to the fact that the activity of mn - sod was synchronized with the conformational changes . the microscopic inactivation rates constants ( ka ) were properly calculated from the semilogarithmic plots ( figure 2(b ) ) where the reactions were plotting as two - phasic courses ( fast , k1 and slow , k2 ) . the rate constants for 0.2 , 0.3 , and 0.4  mm al were obtained as k1 = 7.39 , 7.83 , and 13.3  10  s , respectively , and k2 = 0.58 , 1.25 , and 1.73  10  s , respectively . these results suggested that the mn - sod inactivation by al followed the first - order kinetic process , and the enhancing al concentration could promote the inactivation rate . to compare the enzyme activity changes with the secondary structural changes , we performed the far - uv circular dichroism ( cd ) spectroscopy . as the concentration of alincreased , the overall amount of secondary structure decreased gradually in a dose - dependent manner : specifically , the measurements for both 208 and 222  nm indicated that overall helical contents were decreased with increasing al concentration ( figure 3 ) . interestingly , the overall secondary structure of mn - sod was mostly sustained at lower than 0.3  mm al but the activity was drastically abolished by al in this range as shown in figure 1 . next , tertiary structural changes of mn - sod in the presence of al were also measured by intrinsic and ans - binding fluorescences measurements . the intrinsic fluorescence changes showed that almight induce the unfolding of mn - sod which was monitored by the decrease of intrinsic fluorescence spectra ( figure 4 ) . based on the quenching effect of aland ( 1 ) , we deduced a double reciprocal plot revealing a linear relationship ( figure 5 ) . from this data , we calculated the binding constant as k = 5.4  0.8  10  m and the binding number as n = 1.5  0.3 according to plotting results and ( 1 ) . these results revealed that al has a strong binding affinity for tyrosinase in the absence of substrate and that there are one or two possible binding sites . the kinetics of mn - sod unfolding was also monitored ( figure 6(a ) ) . the data of the semilogarithmic plots showed that the unfolding process also followed the first - order kinetics where the reactions were plotting as two - phasic courses ( fast , ku1 and slow , ku2 ) . the rates constants were calculated as ku1 = 2.89 and ku2 = 0.21  10  s ( figure 6(b ) ) . these results indicated that unfolding process of mn - sod was synchronized with the activity inactivation with the same order reaction , which was comparative to the results in figure 2 . in the next step , we monitored the hydrophobicity changes of mn - sod in the presence of al . the ans - fluorescence intensities were changed by overall range of al concentrations ( figure 7 ) , indicating that hydrophobic surfaces of mn - sod were exposed during al - mediated unfolding . in general , ans dye can bind to hydrophobic amino acid residues , thus , it is used to monitor the tertiary structural disruption of the enzyme in the presence of inactivator . our results showed that increase al concentration caused the mn - sod ans - fluorescence intensity increase in a concentration - dependent manner . because the crystallographic structure of mn - sod from thermus thermophilus has been elucidated ( pdb i d : 3mds ) the docking between mn - sod and al by using dock6.3 was successful with significant score ( 14.07  kcal / mol ) and we searched for al binding residues of mn - sod . we found that the most important expected binding residues interacting with al were asp152 and glu157 residues ( box in figure 8) . the docking simulation provided the supportive information for the inactivation of mn - sod by al where the binding site is not located in the manganese - containing active site pocket ( figure 9 ) . we found that al - induced inactivation of mn - sod is not due to the replacement of manganese or chelating from the active site . several biological effects of al have been reported [ 1215 ] : the results were mostly focused on the toxic effects such as the involvement of oxidative stress , deregulation of cell signaling , membrane biophysics alterations , and the neurotoxicity in neurotransmission . on the contrary , a study reported that al can promote faster wound healing in response to skin injury   when it is prepared as the template to generate large uniform membranes with differing nanopore sizes . our investigation suggested a possible cytotoxic effect of al induced by mn - sod inhibition and it may sequentially induce the oxidative damage . our data consistently supports the previous report that al acted as a toxic material for skin fibroblasts . the changes of enzyme activity and structure in various ions have been extensively studied . for the case of al , it has been applied to several enzymes [ 1820 ] to test its effects on the activities . the biological effect of al has been gradually elucidated and in this regard , we mainly focused on the changes in mn - sod activity and structures in al solutions in the present study , and we found that al worked as an inactivator to mn - sod accompanying with kinetic unfolding processes both in activity and structures . the relative activity and the activity of mn - sod was conspicuously observed when the secondary structure change has not yet occurred . the tertiary structural change of mn - sod by al was confirmed by the result of exposing the hydrophobic surface . even at low concentration of al , the overall structure of mn - sod was changed and this directly affected the structural shape of active site pocket , regardless of mn - sod active center site is very impact and stable due to manganese contained inside . al did not directly compete with substrate but it affect the catalysis with a dose - dependent manner , implying that al binding site is near to substrate docking site . the computational simulations supported this observation that al can form a ligand - binding complex directly with asp152 and glu157 residues of mn - sod , and these amino acid residues   are not located in the active site . therefore , the conformational changes were observed firstly prior to the occurrence of activity loss since the active site is relative compact and stable via manganese presence and al docking site is relative flexible part of mn - sod . according to the results observed in the present study , we deduced the mechanisms of mn - sod response to al : ( i ) al ligand - binding to mn - sod causes fist - order kinetic inactivation which was synchronized with conformational changes ; ( ii ) al also induced the decrease of secondary structure at relatively high concentration but compared to the activity and tertiary structural changes , the secondary structure was less sensitive to al than the tertiary structure ; ( iii ) interestingly , the result of computational simulation support our supposition that mn - sod was bound to the near of active site , not in the inner site of active site pocket . in conclusion , mn - sod from extremophile such as thermus thermophilus was tend to be very stable against the changes of temperature and ph , as well as denaturants addition such as urea and guanidine hydrochloride . however , we found that mn - sod from thermus thermophilus was conspicuously denatured by al . taken together , the inhibition kinetics combined with the computational prediction allowed us to elucidate into the relationship between enzymatic reaction and structural changes of mn - sod from thermus thermophilus and provides greater insight regarding the folding of mn - sod as well as the cytotoxicity of al .