Ratiometric Zinc Sensing in Biological Systems with ZNP1 Probe

1. Principles of Bioinorganic Chemistry - 2004

2. Zin-Naphthopyr 1, A Tautomeric Probe for Ratiometric Zinc Sensing ZNP1 Chang, et al., Proc. Natl. Acad. Sci. USA2004, 101,1129-1134

3. Characterization of ZNP1 F624/F528 Response Fluorescein-like Em = 528 nm Naphthofluorescein-like Em = 624 nm Kd = 0.55 nM Chang, et al., Proc. Natl. Acad. Sci. USA, 2004, 101,1129-1134

4. Ratiometric Imaging in Live Mammalian Cells ZNP1 affords ratiometric Zn2+ sensing in live mammalian cells ZNP1-Ac (20 M) + Zn(pyrithione)2 (50 M) + TPEN (100 M) COS-7 cells Exc 488 nm, ratio imaging 624 nm/528 nm

5. Ratiometric Imaging of Neuronal [Zn2+] ZNP1 affords ratiometric Zn2+ sensing in live neurons ZNP1-Ac (20 mM) + Zn(pyrithione)2 (50 mM) + TPEN (100 mM) embryonic rat hippocampal neurons, DIV 18 Exc 488 nm, ratio imaging 624 nm/528 nm Chang

6. NO-Induced Release of Intracellular Zn2+ Ratiometric imaging in COS cells, lex = 488 nm, lex ratio 612/526 nm ZNP1-Ac (20 mM) + SNOC (10 mM) + TPEN (2 mM) Chang, et al., Proc. Natl. Acad. Sci. USA, 2004, 101,1129-1134

7. NO-induced Release in Neurons Ratiometric imaging in neurons, lex = 488 nm, lex ratio 612/526 nm 15 min after SNOC 45 min after SNOC Before SNOC embryonic rat hippocampal neurons, DIV 18 20 mM ZNP1-Ac, 20 min, 37 °C 100 mM SNOC, 37 °C Chang

8. Reading Resources General References: • R. S. Drago, “Physical Methods for Chemists,” 2nd ed., • Surfside Scientific Publishers, Gainesville, FL (1977) • L. Que, Jr., Ed., “Physical Methods in Bioinorganic Chemistry, • University Science Books, Sausalito, CA (2000) Methods Described: • Mössbauer Spectroscopy • Electron Paramagnetic Resonance Spectroscopy • XAS • Optical and IR spectroscopy will be covered later; also 5.04

9. Energetics of 57Fe Excited State Formation for Mössbauer Effet Studies http://www.webres.com/mossbauer_detail.html

10. Three Factors that Affect the Resonant Energy • Chemical effects Isomer shift (d) • Quadrupolar interactions Quadrupolar splitting DEQ • Externally applied H Additional splitting

11. Resonance Absorption in 57Fe Mössbauer Spectroscopy quadrupolar splitting

12. Magnetic Splitting of Line in 57Fe Mössbauer Spectroscopy

13. Magnetic Splitting of Line in 63/65Cu EPR Spectroscopy

19. The Research Yard at SLAC