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. Author manuscript; available in PMC: 2012 Mar 15.

Published in final edited form as: Arch Biochem Biophys. 2010 Dec 24;507(2):332–342. doi: 10.1016/j.abb.2010.12.022

(A) PEP titration: Reaction mixtures (1 ml volume) contained: Tris·Cl, pH 8, 20 mM; MgCl₂, 2 mM, EI^Ntr(H356A), 0.5 μM. The baseline fluorescence intensities were: H, 4960; D, 5150. Fluorescence at 340 nm was measured at 37 °C after the indicated additions of PEP. The increase in fluorescence is plotted against the PEP concentration. An incubation mixture without added EI^Ntr showed little fluorescence and no change in fluorescence after addition of PEP (data not shown). H form, squares; D form, circles. The measured fluorescence intensities were expressed as the % increase in fluorescence over the baseline. (B) NPr titration: Reaction mixtures (1 ml volume) contained: Tris·Cl, pH 8, 20 mM; MgCl₂, 2 mM, EI^Ntr(H356A), 0.5 μM. The baseline fluorescence intensities were: H, 5985; D, 4986. Fluorescence at 340 nm was measured at 37 °C after the indicated additions of NPr. The increase in fluorescence is plotted against the NPr concentration. An incubation mixture without added EI^Ntr showed some fluorescence change after addition of NPr (data not shown). NPr has no Trp or Tyr residues but does have 3 Phe residues. The sequence is: MTVKQTVEITNKLGMHARPAMKLFELMQGFDAEVLLRNDEGTEAEANSVIALLMLDSA KGRQIEVEATGPQEEEALAAVIALFNS. The data were corrected for this change due to the NPr fluorescence. H-EI^Ntr, filled squares; D-EI^Ntr, open circles. The measured fluorescence intensities were expressed as the % increase in fluorescence over the baseline. (C) Effect of H-EI^Ntr concentration on the PEP-dependent fluorescence change. Experimental conditions were as described above, except that PEP, where added, was at 2 mM. The EI^Ntr(H356A) concentration was varied, as indicated.

(A) PEP titration: Reaction mixtures (1 ml volume) contained: Tris·Cl, pH 8, 20 mM; MgCl₂, 2 mM, EI^Ntr(H356A), 0.5 μM. The baseline fluorescence intensities were: H, 4960; D, 5150. Fluorescence at 340 nm was measured at 37 °C after the indicated additions of PEP. The increase in fluorescence is plotted against the PEP concentration. An incubation mixture without added EI^Ntr showed little fluorescence and no change in fluorescence after addition of PEP (data not shown). H form, squares; D form, circles. The measured fluorescence intensities were expressed as the % increase in fluorescence over the baseline. (B) NPr titration: Reaction mixtures (1 ml volume) contained: Tris·Cl, pH 8, 20 mM; MgCl₂, 2 mM, EI^Ntr(H356A), 0.5 μM. The baseline fluorescence intensities were: H, 5985; D, 4986. Fluorescence at 340 nm was measured at 37 °C after the indicated additions of NPr. The increase in fluorescence is plotted against the NPr concentration. An incubation mixture without added EI^Ntr showed some fluorescence change after addition of NPr (data not shown). NPr has no Trp or Tyr residues but does have 3 Phe residues. The sequence is: MTVKQTVEITNKLGMHARPAMKLFELMQGFDAEVLLRNDEGTEAEANSVIALLMLDSA KGRQIEVEATGPQEEEALAAVIALFNS. The data were corrected for this change due to the NPr fluorescence. H-EI^Ntr, filled squares; D-EI^Ntr, open circles. The measured fluorescence intensities were expressed as the % increase in fluorescence over the baseline. (C) Effect of H-EI^Ntr concentration on the PEP-dependent fluorescence change. Experimental conditions were as described above, except that PEP, where added, was at 2 mM. The EI^Ntr(H356A) concentration was varied, as indicated.