Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions

The interaction of αA- and αB-crystallins with Cu2+ ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αA- and αB-crystallins and αA- and αB-crystallins modified by peroxynitrite on concentration of Cu2+ ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu2+-binding sites. The microscopic dissociation constant for Cu2+–αA-crystallin complex (Kdiss) was found to be equal to 9.7 µM. For peroxynitrite modified αA-crystallin the Kdiss value is equal to 17 µM. One subunit of native αB-crystallin contains two non-equivalent Cu2+-binding sites. The corresponding microscopic dissociation constants for Cu2+–αB-crystallin complexes (K1 and K2) were found to be equal to 0.94 and 36 µM. For peroxynitrite modified αB-crystallin the K1 and K2 values are equal to 4.3 and 70 µM, respectively.


Specifications table
Biochemistry Specific subject area Human α-crystallin, Peroxynitrite, Fluorescence spectroscopy, Cu 2 + ion Type of data Graphs of protein titration by Cu 2 + -ion How data were acquired The Trp-fluorescence spectra of different α-crystallins with titration of increasing concentrations of Cu 2 + -ions were obtained by a Cary Eclipse fluorescence spectrophotometer. Data format Raw and analyzed Parameters for data collection The measurements of protein samples (0.15 mg mL −1 ) were done at 25 °C in buffer A and the protein samples were titrated with increasing concentrations of Cu 2 + -ions (0-300 μM).

Description of data collection
The Trp-fluorescence spectra of native and peroxynitrite modified proteins were measured between 30 0 and 50 0 nm with excitation at 295 nm using fluorescence spectrophotometer.

Value of the data
• The affinity of native and peroxynitrite modified αAand αB-crystallins to copper ions were characterized. • Modification by peroxynitrite results in the decrease in the affinity of these proteins to copper ions. • The obtained data can be used for interpretation of the effect of copper ions on chaperonelike activity of both native and peroxynitrite modified variants of these proteins. • These data might be of beneficial to clinical researches particularly in the case of patients with diabetes mellitus and during aging which are accompanied with elevation of both copper ions and oxidative stress in the lenticular tissues.

Analysis of data on titration of the protein by the specific ligand obtained by fluorescence method. Theory
Consider the approaches to the analysis of the data on titration of the protein (P) by the specific ligand (L) obtained by fluorescence method. It is assumed that the protein molecule contains n ligand-binding sites ( ). The equilibrium + L L is characterized by the microscopic dissociation constant: where [ ] 0 and [ ] are the total and equilibrium concentrations of ligand-binding sites, [L] 0 and [L] are the total and equilibrium concentrations of the ligand. The fluorescence intensity ( I ) is composed of two terms, one of which is proportional to the free binding sites concentration and other is proportional to L complex concentration [1] : When [L] = 0, the initial value of fluorescence intensity is equal to I 0 : I 0 = α[ ] 0 . When [L] 0 → ∞ , the limiting value of fluorescence intensity is equal to I lim : I lim = β[ ] 0 . Thus, Eq. (2) can be written as follows: Let [P] 0 be the initial molar concentration of the protein. The total molar concentration of ligand-binding sites [ ] 0 is equal to n [P] 0 . Taking into accounts Eqs. (1) and ( 3 ), we can obtain the following expression for fluorescence intensity as a function of total concentrations of the protein and ligand: Given the number of the ligand-binding sites in the protein molecule ( n ) this expression allows determining the microscopic dissociation constant K diss from the titration data. If the n value is unknown, the following approach can be used to check the equivalence of the ligandbinding sites and estimate the value of n . Fluorescence measurements allows us to calculate the degree of saturation of the binding sites by the ligand ( Y ): The expression for K diss acquires the following form: This expression can be transformed to the linear anamorphosis: The [L] 0 (1 -Y )/ Y versus Y plot is schematically represented in Fig. 1 . The slope of the linear dependence is equal to -n [P] 0 . The length cut off on the ordinate axis is equal to ( n [P] 0 + K diss ). Consider the situation when the protein molecule contains two non-equivalent binding sites. The dependence of the degree of saturation Y on the equilibrium ligand concentration [L] has the following form: where K 1 and K 2 are the dissociation constants for the complexes of the ligand with the corresponding binding sites. From this equation the [L] value can be expressed as a function of Y : Taking into account that If it has been established that the protein molecule contains two non-equivalent ligandbinding sites, the determination of the dissociation constants K 1 and K 2 can be carried on using coordinates {[L]; r } where r is a number of the ligand molecules bound the protein molecule from fluorescence data using Eq. (5) . The dependence of r on [L] has the following form: (11)  Table S1 in supplementary materials.  Knowing the n value, we can analyze the dependence on fluorescence intensity on the ligand concentration using Eq. (4) without preliminary estimation of the I lim value. The results of fitting Eq. (4) to the experimental data are shown in Fig. 2 . The following values of parameters I lim and K diss were found: I lim = 178 ± 2 and K diss = 9.7 ± 0.7 μM ( R 2 = 0.9962).  Table S3 in supplementary materials. The dependence of fluorescence intensity of peroxynitrite modified αA-Cry on the concentration of Cu 2 + -ions was analyzed with the assumption that αA-Cry subunit contains two equivalent ligand-binding sites. Eq. (4) was used for this purpose ( Fig.   4 ). The initial concentration of peroxynitrite modified αA-Cry calculated on subunit, [P] 0 , in these experiments was 7.5 μM. The following values of parameters I lim and K diss were found: I lim = 108.8 ± 1.5 and K diss = 17 ± 2 μM ( R 2 = 0.9756).    Table S6 in supplementary materials). When fitting Eq. (11) to the experimental dependence  of r on [L], the following values of constants K 1 and K 2 were obtained: K 1 = 0.94 ± 0.19 μM and K 2 = 36 ± 3 μM ( R 2 = 0.9719).  Table S7 in supplementary materials.

Titration of peroxynitrite modified αB-Cry by Cu 2 + -ions
The dependence of fluorescence intensity of peroxynitrite modified αB-Cry on the concentration of Cu 2 + -ions was analyzed with the assumption that αB-Cry subunit contains two nonequivalent ligand-binding sites. Eq. (11) was used for this purpose ( Fig. 9 ; the experimental The results of fluorescence titration analysis have been used for characterization of the affinity of αAand αB-crystallins and αAand αB-crystallins modified by peroxynitrite to Cu 2 + ions [2] .

Expression and purification of recombinant αAand αB-Crys
The cDNA of human recombinant αAand αB-Cry subunits which cloned into the bacterial vector pET-28b ( + ) was expressed in the BL21 (DE3) strain of Escherichia coli as described previously [3] . The centrifugation of cells which were harvested for 16 h after induction, was done at 50 0 0 × g for 20 min at 4 °C. Then, the bacterial cell pellets were re-suspended in 25 mM Tris buffer, pH 7.2, containing 5 mM EDTA, 10 mM β-mercaptoethanol ( β-ME), 100 mM NaCl and 0.01% NaN 3 (lysis buffer). Then, the mixture was sonicated (five time for 30 s with 60% ultrasonic amplitude using a Bandelin Sonopuls sonicator, Berlin, Germany). The bacterial lysates were centrifuged at 8600 × g for 40 min at 4 °C and the supernatant dialyzed against 50 mM sodium phosphate buffer, pH 6.5. After that, the protein sample was loaded on a DEAE-cellulose (0.8 × 15 cm) anion exchange column which pre-equilibrated with the same buffer at 4 °C. The protein fractions were collected at a flow rate of 1 mL ·min −1 in the presence of linear NaCl gradient 0.05-0.4 M in sodium phosphate buffer with a fraction size of 2 mL. The protein concentration was determined with Bradford assay and the highly purified fractions which assessed by SDS-PAGE (12% acrylamide) were collected and dialyzed overnight at 4 °C. The dialyzed sample (against 25 mM Tris buffer, pH 8.0, containing 0.5 mM EDTA, 10 mM β-ME and 0.01% NaN 3 ) was then applied onto a Q-Sepharose (12.5 × 0.5 cm) anion exchange column which pre-equilibrated with the same buffer at 4 °C. The flow rate and fraction size of this column were fixed similar to the DEAE-cellulose column. The bound proteins were eluted with a 0-0.5 M NaCl gradient. The protein fractions were pooled and dialyzed against 25 mM Tris buffer, pH 8.0, containing 0.1 M NaCl, 0.5 mM EDTA, 10 mM β-ME and 0.01% NaN 3 . The concentrated protein samples were then applied onto a Sephacryl S-300HR gel filtration column (1.5 × 100 cm) that pre-equilibrated with the same buffer (4 °C, flow rate 0.25 mL min −1 , fraction size 2 mL) [4][5][6] . The purity of the recombinant αAand αB-Crys were confirmed with SDS-PAGE (12% gel). At the end, the highly purified protein fractions were collected and dialyzed against double distilled water (ddH 2 O) and stored at −20 °C until further use.

Peroxynitrite modification of recombinant αAand αB-Crys
Synthesis of peroxynitrite was done according to the earlier studies [ 7 , 8 ]. The αAand αB-Crys (2 mg mL −1 ) were incubated in the absence and presence of 7 mM peroxynitrite at room temperature for 30 min. Finally, the incubated solutions were individually dialyzed against ddH 2 O to remove excess peroxynitrite by using dialysis tube (cutoff of 10,0 0 0 Da). This experiment was done in 50 mM sodium phosphate buffer, pH 7.4, containing 10 mM HCO 3 − .

The fluorescence measurement of native and peroxynitrite modified αAand αB-Crys
The Trp-fluorescence spectra of native and peroxynitrite modified αAand αB-Crys (0.15 mg mL −1 ) were obtained between 300 and 500 nm after excitation at 295 nm using a Cary Eclipse fluorescence spectrophotometer [ 3 , 9 ]. The measurements were performed at 25 °C in 50 mM sodium phosphate buffer, pH 7.2 (buffer A) and the protein samples were titrated with increasing concentrations of Cu 2 + (0-300 μM). The slit bandwidths were fixed at 10 nm in both channels. The dependence of fluorescence intensity of different protein samples on the concentration of Cu 2 + -ions was evaluated at 337 nm in the absence and presence of different concentrations of Cu 2 + .

Data analysis
Origin Pro 8.0 SR0 software was used for the calculations. To characterize the degree of agreement between experimental data and calculated values, we used the coefficient of determination R 2 (see [10] ).