1 cm quartz cuvette

Stock solutions of Aβ were prepared by reconstituting the lyophilized peptide in a 1:4.5:4.5 (v/v/v) mixture of 60 mM NaOH, Milli-Q water, and 22.2 mM sodium phosphate (pH 7.5) to yield a nominal Aβ concentration of 1 mg/mL in 10 mM sodium phosphate (pH 7.5). The peptide solution then was sonicated for 1 min in a bath sonicator (model 1510, Branson, Danbury, CT) and filtered through a 30 kDa molecular weight cutoff Microcon centrifugal filter device (Millipore, Billerica, MA) for 15 min at room temperature (RT, 22 °C) at 16000g. The filtrate was collected, and the Aβ concentration was determined by UV absorbance (ε₂₈₀ = 1280 cm⁻¹ M⁻¹) using a 1 cm quartz cuvette (Hellma, Plainview, NY) and a Beckman DU-640 spectrophotometer (Beckman Instruments, Fullerton, CA). All measurements were performed at RT. This protocol results in uniform and reproducible material termed low-molecular weight (LMW) Aβ.^{14 (link)}

Aβ42 and [F10, Y42]Aβ42 were synthesized using solid phase peptide synthesis and Fmoc chemistry on an Applied Biosystems model 433A peptide synthesizer (Foster City, CA, USA), as described previously [3 (link)]. Peptide lyophilizates (200 μ g of approximately 80% peptide by weight) were dissolved in 25 μ L of 60 mM sodium hydroxide (Fisher, Waltham, MA, USA) in water to increase solubility and decrease de novo peptide aggregation [29 (link)]. Immediately thereafter, 112.5 μ L of water and 112.5 μL of 22.2 mM sodium phosphate, pH 7.4, were added, and the solution was sonicated in an ultrasonic water bath (model 1510, Branson Ultrasonics Corp., Danbury, CT, USA) for 1 min at 22°C. The pH of the solution at this stage of peptide preparation is ≈11, which facilitates peptide manipulation by increasing solubility and preventing spontaneous self-association [29 (link)]. Protein concentration was determined at 22°C by UV absorbance (ε₂₇₄=1280 cm⁻¹M⁻¹) using a 1 cm quartz cuvette (Hellma, Plainview, NY, USA) and a Beckman DU-640 spectrophotometer (Beckman Instruments, Fullerton, CA, USA). Peptide concentration was adjusted to 80 μ M using 10 mM sodium phosphate, pH 7.4. This yielded a final pH of 7.4.

The spectra were recorded every 10 min over a period of 24 h on a Cary 60 spectrophotometer at room temperature (22–24 °C) in the spectral range 250–850 nm in 1 cm quartz cuvettes (Hellma). A 500 mM potassium phosphate buffer stock at pH 7.4 was prepared by mixing potassium dihydrogen phosphate 99% (KH₂PO₄) with potassium hydrogen phosphate 98% (K₂HPO₄) in Milli-Q water. Stock solutions were further diluted to the desired 100 mM concentration for the following experiments. 200 mM reduced GSH stock solutions were prepared immediately before the experiment and appropriate portions for the 7:1, 5:1, 3:1, and 1:1 (GSH:Cu) ratios were added to the cuvette containing 1 mM Cu(NO₃)₂ to obtain final GSH concentration of 0.35–2.45 mM and Cu(II) 0.35 mM. Experiments with such different ratios gave qualitatively the same general results, differing only by the Cu(I) reoxidation time, which was correlated with the excess of GSH not bound to Cu(I). The Cu(I) reoxidation to the Cu(II) complex of GSSG was enabled by oxidative exhaustion of GSH by ambient oxygen, as verified by ESI-MS.