Dimethyl sulfoxide buffer solution preparation

To prepare the ninhydrin reagent, ninhydrin (2 g) and hydrindantin (300 mg) are dissolved in dimethyl sulfoxide (DMSO) (75 ml) in a foil-covered 400 ml flask (to exclude light) and are adjusted to 100 ml with 4 M lithium acetate buffer. The jar is stoppered with a rubber bung with inlet and outlet tubes, and the reagent is flushed with 02-free N2 for 30 minutes. The operations must be done in a fume hood. The final solution is then stored sealed at room temperature. Ninhydrin is toxic and stains the skin, so gloves should be worn at all times. 

Stock solutions (20 mol m ) of substituted 9,10-anthraquinones (Aldrich Chemical Company) were prepared in dimethyl sulfoxide or 1,2-dimethoxyethane. Previous studies [7-10] determined that these solvents induced no quenching of chlorophyll fluorescence at the concentrations employed in diluted chloroplast samples. Spinach chloroplasts were isolated as in previous studies [7-10] and suspended in a buffer of 10 mol m sucrose/50 mol m KEPES-NaOH (pH 1.5)/5 mol m NaCl. Fluorescence samples contained [chlorophyll] = 0.010 kg m"" and [quinone] = 0 to 0.200 mol m . Quinones were incubated in chloroplast solutions for 5 minutes at 5 C in the dark to permit diffusion to the site of action [7-10]. 

Reaction buffers were prepared by adding 4 volumes of 10 mM aqueous potassium dihydrogen phosphate odium hydroxide buffer (pH 6.S-7.6) to 6 volumes of dimethyl sulfoxide. The resulting solutions had pHs ranging from 10.6 to 11.5 as determined with the glass electrode. -Cyclodextrin solutions (0.9-6.0 mM) were prepared with this buffer and stored under nitrogen. Substrate solutions (3-4 mM in dimethyl sulfoxide) were stored in the dark. 

Ultrapure water (18.2 MS2 cm), was obtained from a MiUipore water purification system and was used for preparation of sample solutions. Analytical Grade Acetonitrile (MeCN) (Fischer Scientific) and Trifluoroacetic Acid (TEA) (Sigma-Aldrich) were used for all ZipTip buffers (see Sect. Denaturing MS— ZipTip Buffers ). The coenzyme A derivatives (acetyl-, butyryl- and malonyl) used in assays were purchased from Sigma-Aldrich. iV-acetylcysteamine (SNAC) thioesters were provided by the Piel lab (ETH, Zurich), and stock solutions were solubilised in dimethyl sulfoxide (DMSO) (Fischer Scientific). lodoacetamide (Sigma-Aldrich) was used for all alkylation reactions. 

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