Novel derivatization of protein thiols with fluorinated fluoresceins

Tetrahedron Letters

Volumn 37, Issue 44, 1996, Pages 7905-7908

  Gee, Kyle R ^a   Sun, Wei Chuan ^a   Klaubert, Dieter H ^a   Haugland, Richard P ^a   Upson, Rosalyn H ^a   Steinberg, Thomas H ^a   Poot, Martin ^a  

^a MOLECULAR PROBES INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; DERIVATIZATION; FLUORESCENCE; PROTEIN ANALYSIS; REACTION ANALYSIS;

STAPHYLOCOCCUS PHAGE 3A;

EID: 0030605055     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/0040-4039(96)01794-7     Document Type: Article

References (16)

1. 1. Lundblad, R.L.; Noyes, C.M., Chemical Reagents for Protein Modification, Vol. I, CRC Press, New York, 1984.
2. 2. Brinkley, M. Bioconjugate Chemistry 1992, 3, 2-13.
3. 3. Corrie, J.E.T; Craik, J.S. J. Chem. Soc. Perkin Trans. I 1994, 2967-2973.
4. 4. For examples of recent efforts, see: a) Langmuir, M.E.; Yang, J.-R.; Moussa, A.M.; Laura, R.; LeCompte, K.A. Tetrahedron Letters 1995, 36, 2989-2992;
5. b) Walker, M.A. J. Org. Chem. 1995, 60, 5352-5355;
6. c) Corrie, J.E.T. J. Chem. Soc. Perkin Trans. 1 1994, 2975-2982.
7. 5. Bolton, R.; Sandall, J.P.B. J. Chem. Soc. Perkin Trans. II 1978, 1288-1292, and references cited therein.
8. 4: C, 59.42; H, 1.99. Found: C, 59.39; H, 2.01. Resorcinol and 4-chlororesorcinol are commercially available. For a synthesis of 4-fluororesorcinol, see Patrick, B.T.; Darling, D.L. J. Org. Chem. 1986, 51, 3242-3244.
9. 2O: C, 49.82; H, 2.09. Found: C, 49.56; H, 2.32. The reaction proceeded more slowly in acetonitrile.
10. 7: C, 59.03; H, 2.48. Found: C, 59.00; H, 2.69.
11. -1 at 515 nm for the free dye (1a).
12. 10. E. coli β-galactosidase contains 64 cysteine residues/enzyme, (Craven, G.R.; Steers, E.; Anfinsen, C.B. J. Biol. Chem. 1965, 240, 2468-2477) with varying amounts oxidized as cystine bridges, depending on preparation and purification methods. According to the manufacturer, the lots used for the experiments described herein were determined to contain 16.0 free sulfhydryl groups/molecule. Titration with thiol reagents has demonstrated that modification of a large number of cysteinyl residues occurs without loss of enzyme activity (Wallenfels, K.; Müller-Hill, B.; Dabich, D.; Streffer, C.; Weil, R. Biochem. Z. 1964, 340, 41-55).
13. 10. E. coli β-galactosidase contains 64 cysteine residues/enzyme, (Craven, G.R.; Steers, E.; Anfinsen, C.B. J. Biol. Chem. 1965, 240, 2468-2477) with varying amounts oxidized as cystine bridges, depending on preparation and purification methods. According to the manufacturer, the lots used for the experiments described herein were determined to contain 16.0 free sulfhydryl groups/molecule. Titration with thiol reagents has demonstrated that modification of a large number of cysteinyl residues occurs without loss of enzyme activity (Wallenfels, K.; Müller-Hill, B.; Dabich, D.; Streffer, C.; Weil, R. Biochem. Z. 1964, 340, 41-55).
14. 11. Human B-cells were cultured in RPMI 1640 medium suplemented with 10% PBS, 100 U penicillin, 100 μg streptomycin and 300 μg L-glutamine per mL of culture medium. Stock solutions of 3 in DMSO were added to cell culture medium to obtain a final dye concentration of 1 μM. The cells were incubated at 37 °C for 30 min, centrifuged, then the staining solution was discarded. The pellets were gently resuspended in PBS and incubated for 15 min, then centrifuged again and resuspended in either fresh PBS solution (for cells to be lysed) or in warm 3.7% formaldehyde in PBS for fixation. The cells in formaldehyde were incubated twice for 10 min, centrifuged, and resuspended in fresh culture medium for flow cytometry using a FACS™-Vantage instrument equipped with an argon-ion laser (488 nm excitation); fluorescence emission was collected using a 515 nm longpass filter and a single photomultiplier tube. The sheath fluid was 0.9% NaCl, and typical sample flow rates were between 200 and 400 particles per second.
15. 12. For gel electrophoretic analysis, labeled cells in PBS were heated briefly with SDS gel loading buffer at 90°C, then allowed to cool and loaded onto 15% (37.5:1) polyacrylamide, 0.05% SDS gels. After electrophoresis, gels were placed directly on a UV transilluminator (300 nm) and photographed. Subsequently, the gels were stained with SYPRO® Orange, a fluorescent protein gel stain, to confirm that the bands labelled with 3 were indeed high molecular weight components and not small molecule thiols such as glutathione (Steinberg, T.H.; Jones, L.J.; Haugland, R.P.; Singer, V.L. Anal. Biochem. 1996, 239, 223-237; Steinberg, T.H.; Haugland, R.P.; Singer, V.L. Anal. Biochem. 1996, 239, 238-245).
16. 12. For gel electrophoretic analysis, labeled cells in PBS were heated briefly with SDS gel loading buffer at 90°C, then allowed to cool and loaded onto 15% (37.5:1) polyacrylamide, 0.05% SDS gels. After electrophoresis, gels were placed directly on a UV transilluminator (300 nm) and photographed. Subsequently, the gels were stained with SYPRO® Orange, a fluorescent protein gel stain, to confirm that the bands labelled with 3 were indeed high molecular weight components and not small molecule thiols such as glutathione (Steinberg, T.H.; Jones, L.J.; Haugland, R.P.; Singer, V.L. Anal. Biochem. 1996, 239, 223-237; Steinberg, T.H.; Haugland, R.P.; Singer, V.L. Anal. Biochem. 1996, 239, 238-245).