-amyloid ( a ) peptide is the main cause of alzheimer 's disease ( ad ) [ 1 , 2 ] . a has two major types , a40 and a42 , which are derived from a ubiquitous type i transmembrane protein amyloid precursor protein ( app ) by a two - step secretase ( - and -secretase ) pathway [ 35 ] . the amyloid cascade hypothesis predicts that the aggregated a peptides in the brain have an early and essential role in the neuronal degeneration that leads to dementia [ 1 , 2 , 6 ] . the aggregation of a occurs following a conformational conversion from either -helix or random coil to -sheet in a time - dependent manner , indicating that the formation of -sheet structure is the key step for the peptide aggregation [ 7 , 8 ] . the formation of a aggregates is modulated by several factors , including metal ions [ 9 , 10 ] . in amyloid plaques of ad - affected brain , remarkably high concentration of metals , such as cu ( 400  m ) , zn ( 1  mm ) and fe ( 1  mm ) , has been found [ 11 , 12 ] . in vitro studies , micromolar levels of cu , zn and fe have been shown to sufficiently induce protease - resistant aggregation and precipitation of a . copper ions are bound to the three his residues of a located at the n - terminus , which forms a 3n1o square - planar motif as verified by epr spectroscopy . copper - selective chelators can dissolve a deposits extracted from ad postmortem brain specimens and reduce the level of free radical . curtain and his colleagues previously reported that the addition of cu to oligomeric a40 in a negatively charged lipid membrane can induce a peptides reinserted into lipid membranes , convert a conformation from -strand into -helix , and cause the lipid peroxidation . their result is the first study to show that instead of -sheet , a in -helical form can also generate free radicals in the presence of cu ions . recently , our group has characterized the conformation of a in the presence of cu ions into three structural states , stable helix , unstable helix , and random coil , in 40% , 25% , and 5% tfe , respectively , . therefore , this provides us a possibility to re - examine the relationship between a conformation , and cu - driven free radical generation . in the present study , we examined the correlation between cu binding affinity , h2o2 formation , and a40 conformation . it was found that both cu binding affinity and h2o2 formation are well correlated with the conformation of a40 . in general , cu ions can be bound to the different structural forms of a40 . the copper - binding affinity of a40 in random coiled form is about 3-fold higher than that of a40 in stable helical form . for h2o2 formation , our result shows in the first time that the generation of h2o2 can also be induced by cu and the stable helical form of a40 . in contrast , in random coiled form , the formation of h2o2 is inhibited by a40 . the synthesis of full - length a40 peptide was performed in an abi-433a solid - phase peptide synthesizer using the fmoc protocol . cleavage and deprotection of the synthesized peptide were performed by treatment with a mixture of trifluoroacetic acid / distilled water / phenol / thioanisole / ethanedithiol . then the peptide was extracted with 1  :  1 ( v  :  v ) ether  :  h2o containing 0.1% 2-mercaptoethanol . the synthesized a peptides were purified on a reverse - phase c-18 hplc with a linear gradient from 0% to 100% acetonitrile , and the molecular weight of a40 peptides and purity was verified by an maldi - tof mass spectroscopy . one mg of purified monomeric a40 peptide was dissolved in 1  ml 100% trifluoroethanol ( tfe ) as stock solution . a final a peptide concentration of 30  m in either 5% or 40% tfe was prepared from fresh peptide stock solution diluted in phosphate buffer , ph 7.4 , with or without the same concentration of cu . a thousand m of cu stock solution was prepared by dissolved cucl2 salt in distilled h2o purged by n2 in a sealed flask and freshly diluted into the designed concentration before it was used . all measurements were performed in quartz cells with a path length of 0.1  cm . data were collected at wavelengths from 190 to 260  nm in 0.2  nm increments . the reported cd spectra were corrected using phosphate buffer , ph 7.4 for the baseline . an equal molar 300  m of a and cu in 30% glycerol phosphate buffer , ph 7.4 , and 5% and 40% of tfe was employed for spin counting purposes . epr spectra were obtained at x - band using a bruker emx er073 spectrometer equipped with a bruker te102 cavity and an advanced research system continuous - flow cryostat ( 4.2300  k ) . during epr experiments , the sample temperature was maintained at 10  k. the microwave frequency was measured with a hewlett - packard 5246l electronic counter . tyrosine fluorescence spectroscopy was used to investigate the binding affinity for copper bound to a. a peptide stock solution was diluted in phosphate buffer , ph 7.4 to a final peptide concentration of 10  m with designed concentration of cu . all measurements were performed in a 96-well plate using a microplate reader ( flexstation 3 , md ) . the excitation and emission wavelengths were selected at 278 and 305  nm , respectively . reported data were the average obtained from three individual samples and three repeated measurements of each sample . the binding affinity was calculated using the following equation : 
 ( 1)fxf0=ixii0i=11+ka[cu2+]n , 
							 where i0 and ix are the fluorescence intensity for free and a/cucomplex , respectively , i is the fluorescence intensity at saturation state , [ cu ] is the copper concentration , and n is the copper binding number . the related parameters were fitted using nonlinear curve fitting program micro - origin v6.0 ( microcal software , inc . , nothampton , ma ) . in the initial fitting stage , the simplex method , which was set to 100 cycle runs , the final fitting parameters were obtained when the value of  was less than 0.05 and the parameters and errors for the parameters reached the convergent and steady state . the production of h2o2 was analyzed using the dichlorofluorescein diacetate ( dcfh - da ) assay . dichlorofluorescein diacetate was dissolved in 100% dimethyl sulfoxide , deacetylated with 50% v / v 0.05 m naoh for 30  min , and then neutralized ( ph 7.4 ) to a final concentration of 200  m as stock solution . the reactions were carried out in dulbecco 's pbs , ph 7.4 , in a 96-well plate ( 100  l / well ) containing different concentration of a40 , 30  m cucl2 , tfe ( 5% and 40% ) , deacylated dcf ( 20  m ) , and horseradish peroxidase ( 5  m ) at 37c . for negative control , an extra of 1.0  mm catalase ( aspergillus niger ) was used to quench h2o2 . fluorescence readings were recorded on a microplate reader ( md , flexstation 3 ) , with the excitation and emission wavelengths selected at 485 and 530  nm , respectively . uv / vis turbidity assay was used to detect the aggregation process of a. 200  l of 30  m a40 in phosphate buffer , ph 7.4 containing either 5% or 40% tfe was freshly prepared from stock solution . fresh prepared samples with or without 30  m cu placed in a 96-well plate were incubated at 37c . turbidity was measured using a microplate reader ( md , flexstation 3 ) at a wavelength of 450  nm . the aim of this study is to examine the relationship between a40 conformation and other physical and chemical properties such as aggregation , cu - binding affinity , and free radical formation . in the present study , we applied 5% and 40% tfe to mimic the conformation of a40 in random coil and stable helix , respectively , . therefore , the effect of tfe on cu binding affinity and free radical generation has to be evaluated . as depicted in supplemental data ( see supplementary material available online at doi:10.4061/2011/607861 ) , the main effect of tfe on the system is only to quench the dcf fluorescence intensity . neither was the solubility of cu ions nor the formation of h2o2 affected by tfe . first of all , we characterized the structural state of a40 with and without cu in either 5% or 40% tfe using circular dichroism ( cd ) spectroscopy . figures 1(a ) and 1(b ) show the cd spectra for a40 and a40/cu in 5% and 40% tfe , respectively . it can be seen that , in 5% tfe , the pattern of cd spectrum for a40 in the presence of cu showed a dramatic change compared to that for a40 only , while the cd spectra of a40 with or without cu in 40% tfe did not show any significant change . the secondary structure of a40 in 5% tfe changes from 63% random coil , 34%  -sheet and 3% helix in cu - free state to 45% random coil , 50%  -sheet and 5% helix in cu - bound state , representing that there is a structural conversion from random coil into -sheet . on the other hand , the conformation of a40 with or without cu in 40% tfe remains stable in which the secondary structure is 74% helix , 14%  -sheet , and 12% random coil in cu - free state and 73% helix , 15%  -sheet , and 12% random coil in cu - bound state , indicating that there is no conformational conversion of a40 in 40% tfe while adding cu ions . it has been shown that cu ions are able to accelerate the aggregation of a40 [ 9 , 15 , 18 , 20 ] . hence , we characterized the aggregation process of a40/cu and a40 in 5% and 40% tfe . the aggregation process of a40/cu and a40 in 5% and 40% tfe was analyzed by turbidity assay as shown in figure 2 . in general , in both 5% and 40% tfe , a40 in the presence of cu was aggregated much faster than that in the absence of cu furthermore , the aggregation rate in 5% tfe for both a40/cu and a40 was much faster than that in 40% tfe . the aggregation process in 5% tfe for a40/cu and a40 showed a typical sigmoidal profile . on the other hand , the aggregation process in 40% tfe for a40 with and without cu mostly stayed in the nucleation or the early elongating stage within 24  hrs . the aggregation process for a40 and a40/cu in 5% and 40% tfe echoes with the result obtained in the analyses of secondary structure in which the aggregation ability is correlated with the ability of conformational formation of -sheet . in the present study , we showed that the conformational states for a40/cu and a40 in 5% are dramatically different , while no such conformational change can be observed between a40/cu and a40 in 40% tfe . although cu ions have been shown to be bound to a in aqueous solution , then , whether cu ions are bound to a40 in 40% tfe is needed to be investigated since the structural state for a40/cu and a40 in 40% tfe shows no difference . to address this issue , epr spectroscopy was applied to explore the interaction between cu and a40 in 40% tfe . as shown in figure 3 , the epr spectra of a/cu both in 5% and 40% tfe showed two major groups of hyperfine peaks , while no hyperfine peak could be observed in the epr spectrum for cu alone , indicating that cu ions were bound to a40 in both 5% and 40% tfe . the pattern of hyperfine peaks for a40/cu in 5% and 40% tfe is similar , but the position of hyperfine peaks in 40% tfe shifts to low magnetic field . the related epr parameters of g , a , and g are 2.28 , 156.8 , and 2.06 for a40/cu in 5% tfe , respectively . the g and g values in 5% tfe are very close to those in the literature measured in aqueous condition , representing that the binding geometry of a40/cu in 5% tfe should adopt a similar 3n1o coordination . on the other hand , the related epr parameters of g , a , and g are 2.40 , 119.6 , and 2.09 for a40/cu in 40% tfe , indicating that the copper - binding geometry is different from the 3n1o mode and this further indicates that the binding affinity of cu to a40 in random coiled form may be stronger than that to a40 in stable helical form . as shown in epr studies , cu can be bound to different structural forms of a40 with a different binding affinity . in order to further elucidate the binding property of cu , we applied the tyrosine fluorescence spectroscopy to determine the cu - binding constant , since tyrosine 10 is the only fluorophore in a amino acid sequence and locates at the binding pocket , in which the binding of cuto a40 may cause the change of tyrosine fluorescence . the titration curves of cu concentration versus tyrosine fluorescence intensity in 5% and 40% tfe are shown in figures 4(a ) and 4(b ) , respectively . the binding constants , ka , were estimated using the equation as described in the experimental section with molar ratio of a  :  cu = 1  :  1 , that is , n = 1 . the calculated ka values are 0.14  m ( r > 0.97 ) and 0.05  m ( r > 0.96 ) for a/cu in 5% and 40% tfe , respectively . the ka of a40/cu in 5% tfe is around 3-fold higher than that in 40% tfe , suggesting that cu is bound to random coiled form of a40 more strongly than to stable helical form of a40 which is consistent with the epr studies . it has been demonstrated that a40 coordinated with cu can cause the reduction of cu and then induce the formation of h2o2 from o2 in a catalytic manner [ 15 , 20 ] . to examine the relationship between free radical formation and a40 conformation , figures 5(a ) and 5(b ) show the plots of dcf fluorescence intensity versus a concentration in 5% and 40% tfe as incubated at 37c for 1  hr . in general , results clearly show that the formation of h2o2 is also correlated with a40 conformation . in 5% tfe , the level of h2o2 was reduced to zero when the a40/cu molar ratio 1 , indicating that the formation of h2o2 is inhibited when a40 adopts the random coiled form . unexpectedly but interestingly , unlike the formation of h2o2 which was inhibited in 5% tfe , the level of h2o2 in 40% tfe was increased with an increase of a40 concentration and was much higher than those of either cu or a40 alone , suggesting that the formation of h2o2 was induced when a40 was in the stable helical form . according to amyloid cascade hypothesis , aggregated a is the main toxic species to cause the alzheimer 's disease . the various forms of a aggregate have been shown to coordinate with redox active transition metals , such as cu and fe , and induce the generation of reactive oxygen species [ 15 , 20 ] . however , the correlation of copper - binding affinity , copper - driven aggregation , and free radical formation with a40 conformation still needs to be elucidated .   for ion interaction , our results demonstrate for the first time that cu ions can interact with both random coiled and stable helical forms of a40 . as a larger a value and binding constant obtained for a40 in 5% tfe , the cu - binding affinity for random coiled form of a40 is much stronger than that for stable helical form of a40 . the possible explanation may be attributed to the different flexibility of a40 conformation in 5% and 40% tfe . as shown in cd spectra , the conformation of a40 in 5% tfe is relatively flexible and can easily convert structure from random coil into -sheet while coordinating with cu . on the other hand , the overall conformation of a40 with or without cu is relatively rigid and remains in stable helix in 40% tfe , and only the local geometry of cu - binding site is distorted while interacting with cu ions as indicated by the shift of g value . therefore , the interaction between cu and a40 is strong in 5% tfe and relatively weak in 40% tfe . the binding affinity for stable helical form of a40 is almost 3-fold lower than that for random coiled form of a40 . the cu - binding affinity can further be used to account for the differences for cu - driven aggregation between random coiled and stable helical forms of a40 . it is well known that the formation of -sheet plays a key step for the aggregation of a40 . as shown in the present study , the conformation of a40 in 5% tfe is much easier than that in 40% tfe to convert into -strand structure by cu ions . thus , in the presence of cu , the random coiled form of a40 ( in 5% tfe ) aggregates faster and more severe than the stable helical form of a40 ( in 40% tfe ) .   unlike the cascade hypothesis that only -sheet form of aggregated a can produce free radical , we demonstrate that helical form of a40 can also induce the formation of h2o2 . from the aggregation assay , this helical form of a40 may possibly exist as a monomer during the early incubation period . then , this further implies that monomeric a40 in stable helical form by coordinating with cu can induce the formation of h2o2 . in contrast , a40 in random coiled form shows to inhibit the formation of h2o2 during the early incubation period . the inhibition of h2o2 formation becomes more significant with an increase of a40 concentration , and the formation of h2o2 further is completely inhibited when the molar ratio of a/cu molar ratio 1 . our observation is consistent with a recent study by viles and his colleagues , in which they showed that , at a/cu molar ratio = 1 , the formation of h2o2 is inhibited by both monomeric and fibrillar a peptides . taken together , it may suggest that helical structure may play a key factor for the generation of h2o2 by a40/cu . in summary , our present results demonstrate that both ion interaction and copper - driven free radical formation are well correlated with a40 conformation . when a40 adopts a stable helical structure , the cu binding affinity is weaker , and the free radical is produced . on the other hand , when a40 adopts a random coil in 5% tfe , the cu binding affinity is stronger , and the formation of h2o2 is inhibited .