Amersham ultrospec 2100 pro

Activity of glyoxalase I (Glo-I, E.C.4.4.1.5) was determined by measurement of the reaction intermediate S-D-lactoylglutathione with ascending absorbance at 240nm [13 ]. Absorbance was measured for 5 min at 25°C in crystal cuvette (Hellma, Berlin, Germany) at photometer (amersham ultrospec 2100 pro, amershampharmacia biotech, Cambridge, England). For each test 2mM GSH (Roth, Karlsruhe, Germany) and 2mM MGO (Sigma-Aldrich, Steinheim, Germany) were incubated for 90 sec in 50mM phosphate-buffer (Na₂HPO₄, pH 7.0, Roth, Karlsruhe, Germany) and 10μl of undiluted cell lysate were used per test. Each probe was measured three times. Phosphate-buffer was set as reference. Enzyme activity was calculated in U by formula: A = (ΔE/min x V) / (ε x d x v). ε for S-D-lactoylglutathione was 2.86 (mol/l x cm). For specific activity U were referred to protein-concentration.

Activity of glyoxalase I (Glo-I, E.C.4.4.1.5) was determined by measurement of the reaction intermediate S-D-lactoylglutathione, with ascending absorbance at 240 nm. Absorbance was measured for 5 min at 25°C in crystal cuvette (Hellma, Berlin, Germany) with a photometer (amersham ultrospec 2100 pro, amershampharmacia biotech, Cambridge, England). For each test, 2 mM GSH (Roth) and 2 mM MGO (Sigma-Aldrich) were incubated for 90 s in 50 mM phosphate-buffer (Na₂HPO₄, pH 7.0, Roth), and 10 μl of undiluted cell lysate were used per test. Each probe was measured three times. Phosphate-buffer was set as reference. Enzyme activity was calculated in U by formula: A = (ΔE/min × V)/(ε × d × v). ε for S-D-lactoylglutathione was 2.86 (mol/l × cm). For specific activity, U was referred to protein-concentration (23 (link)).