Materials

2.1. Generation of 1. HEN buffer for protein extraction: 25 mM N-(2-

S-Nitrosothiols hydroxyethyl)piperazine-N -2-ethanesulfonic acid (HEPES)-

NaOH, pH 7.7; 1 mM ethylene diamine tetraacetic acid (EDTA); and 0.1 mM neocuproine (Sigma). Buffers containing neocuproine should be protected from light.

250 |JM 250 |JM HjO NO untreated GSNO SNP

Fig. 15.2. Nitrosothiol content in Arabidopsis thaliana cell suspension culture extracts after treatment with NO donors and in leaves of NO-treated A. thaliana plants. After treatment of cell culture extracts with either 250 \M of GSNO, 250 \M of SNP, or water, remaining NO donors were removed by the use of chromatography on a Sephadex G-25 M column. Nitrosothiol contents were determined according to Saville (29). Additionally, nitrosothiol content from leaves of A. thaliana plants treated with NO gas and from leaves of untreated plants were measured. Treatment of cell culture extracts with 250 \M of GSNO resulted in a nitrosothiol content of approx 40 \M/mg protein. The nitrosothiol content in Arabidopsis leaves treated with NO gas reached values up to 30 \M/mg protein. Values represent mean of at least two independent determinations .

250 |JM 250 |JM HjO NO untreated GSNO SNP

Fig. 15.2. Nitrosothiol content in Arabidopsis thaliana cell suspension culture extracts after treatment with NO donors and in leaves of NO-treated A. thaliana plants. After treatment of cell culture extracts with either 250 \M of GSNO, 250 \M of SNP, or water, remaining NO donors were removed by the use of chromatography on a Sephadex G-25 M column. Nitrosothiol contents were determined according to Saville (29). Additionally, nitrosothiol content from leaves of A. thaliana plants treated with NO gas and from leaves of untreated plants were measured. Treatment of cell culture extracts with 250 \M of GSNO resulted in a nitrosothiol content of approx 40 \M/mg protein. The nitrosothiol content in Arabidopsis leaves treated with NO gas reached values up to 30 \M/mg protein. Values represent mean of at least two independent determinations .

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