pH-Dependent Synthesis of Anisotropic Gold Nanostructures by Bioinspired Cysteine-Containing Peptides

In the present study, alkaline peptides AAAXCX (X = lysine or arginine residues) were designed based on the conserved motif of the enzyme thioredoxin and used for the synthesis of gold nanoparticles (GNPs) in the pH range of 2–11. These peptides were compared with free cysteine, the counterpart acidic peptides AAAECE and γ-ECG (glutathione), and the neutral peptide AAAACA. The objective was to investigate the effect of the amino acids neighboring a cysteine residue on the pH-dependent synthesis of gold nanocrystals. Kohn–Sham density functional theory (KS-DFT) calculations indicated an increase in the reducing capacity of AAAKCK favored by the successive deprotonation of their ionizable groups at increasing pH values. Experimentally, it was observed that gold speciation and the peptide structure also have a strong influence on the synthesis and stabilization of GNPs. AAAKCK produced GNPs at room temperature, in the whole investigated pH range. By contrast, alkaline pH was the best condition for the synthesis of GNP assisted by the AAARCR peptide. The acidic peptides produced GNPs only in the presence of polyethylene glycol, and the synthesis using AAAECE and γ-ECG also required heating. The ionization state of AAAKCK had a strong influence on the preferential growth of the GNPs. Therefore, pH had a remarkable effect on the synthesis, kinetics, size, shape, and polydispersity of GNPs produced using AAAKCK. The AAAKCK peptide produced anisotropic decahedral and platelike nanocrystals at acidic pH values and spherical GNPs at alkaline pH values. Both alkaline peptides were also efficient capping agents for GNPs, but they produced a significant difference in the zeta potential, probably because of different orientations on the gold surface

Oxidative modifications induced by a TR cation radical on cytochrome

<p><b>c</b>.A - Spectral changes of cytochrome <i>c</i> before (light gray line) and after 30 (gray line), and 120 (black line) min UV light irradiation at 254 nm, at pH 4.0. <b>B</b> –The same conditions described for A in the presence of 25 µmol/L TR. <b>C</b> - The same conditions described for Ain the presence of 2.5 mmol/L TR. In this panel, the dashed line represents the original spectrum of cytochrome c before the subtraction of the turbidity sample contribution that resulted in the spectrum shown as a black line <b>D</b> – The effect of different TR concentrations on the rate of Soret band blue shift promoted by UV irradiation. In panel D, the number of asterisks indicates the corresponding spectra shown in panel A (*), B (**), and C (***), from which the data were extracted. The inset shows SEM images and the distribution of particle size obtained for the sample in the condition marked with three asterisks. <b>E</b> – The effect of pH on the rate of Soret band blue shift promoted by UV irradiation in the presence of 25 µmol/L TR. Filled black circles represent the results in the absence of TR and opened circles represent the presence of TR. In panel E, the asterisk in one data point indicates that the point was extracted from the corresponding spectrum shown in panel F. <b>F</b> – The same conditions described for B, at pH 8.0.In this panel, the dashed line represents the original spectra of cytochrome c before the subtraction of the turbidity sample contribution that resulted in the spectrum shown as a black line. Samples containing 3 µmol/L cytochrome <i>c</i> and TR, when indicated, were irradiated in a 5 mmol/L phosphate buffer at 25°C in the cuvette with a 4 mW/cm^-2 as function of 254 nm of excitation at a distance of 4 cm from 1cm² of a selected sample area.</p

Oxidative modifications induced by FP cation radicals on cytochrome

<p><b>c</b>.A - Spectral changes of cytochrome <i>c</i> before (light gray line) and after 30 (gray line), and 120 (black line) min UV light irradiation at 254 nm in the presence of 10µmol/LFP, at pH 4.0. <b>B</b> - The same conditions described for A in the presence of 2.5 mmol/LFP. In this panel, the dashed line represents the original spectra of cytochrome <i>c</i> before the subtraction of the turbidity sample contribution that resulted in the spectrum shown as a black line <b>C</b> – The effect of different FP concentrations on the rate of Soret band blue shift promoted by UV light irradiation. In panel C, the number of asterisks indicates the corresponding spectra shown in panel A (*) and B (**), from which the data were extracted. The inset shows SEM images and the distribution of particle size obtained for the sample in the condition marked with two asterisks. <b>D</b> – The effect of pH onthe rate of Soret band blue shift promoted by UV light irradiation promoted by 10 µmol/L FP. Filled black circles represent the results in the absence of FP and opened circles represent the presence of FP.In panel E, the asterisk in one data point indicates that was the point extracted from the spectra shown in panel E. <b>E</b> – The same conditions described for B, at pH 8.0. In this panel, the dashed line represents the original spectra of cytochrome <i>c</i> before subtraction of the turbidity sample contribution that resulted in the spectrum shown as a black line. Samples containing 3 µmol/Lcytochrome <i>c</i> and FP, when indicated, were irradiated in a 5 mmol/Lphosphate buffer at 25°C in the cuvette with a 4 mW/cm^-2of UV light intensity as function of 254 nm of excitation at a distance of 4 cm from 1cm² of selected sample area.</p

EPR and MCD analysis of cytochrome c modified by phenothiazinesA

<p>- EPR spectra of native cytochrome c; <b>B</b> - EPR spectra of cytochrome <i>c</i> 120 min irradiation in the presence of TR⁺. The spectra were obtained in the presence of 100 µmol/L cytochrome <i>c</i> and 200 µmol/L TR⁺ in a 5 mmol/L phosphate buffer, pH 7.4, at 11 Kelvin, 1 mT of field modulation, and 4 mW of microwave power in the X band. <b>C</b> – EPR spectra of TR at room temperature, which were obtained in the same conditions described for panel B. In this panel, the light gray line represents the spectrum obtained in the absence of cytochrome <i>c</i> and the black line the spectrum obtained in the presence of cytochrome <i>c</i>. Samples containing cytochrome <i>c</i> and TR were irradiated during 120 min in a 5 mmol L^-1phosphate buffer, at 35°C in the cuvette with 4 mW/cm^-2 of UV light intensity as function of 254 nm of excitation at a distance of 4 cm from 1cm² of selected sample area. <b>D</b> - MCD spectra of cytochrome <i>c</i> obtained after 120 min of irradiation in the presence of TR (gray line). In this panel, the dashed line represents the spectrum of an identical concentration of cytochrome <i>c</i> (100 µmol/L), completely converted to the ferrous form by β-mercaptoethanol. The samples were irradiated, with 4 mW cm^-2 of UV light intensity as function of 254 nm of excitation at a distance of 4 cm from 1cm² of selected sample area, in 10 mmol/L universal buffer, pH 5.</p