Hydrazide fluorescein-5-thiosemicarbazide

Ahn, B., Rhee, S. G., and Stadtman, E. R. (1987) Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Anal. Biochem. 161,245— 257. 

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of fluorescein that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 208) (Pierce). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment, the compound has an extinction coefficient of approximately 78,000 M-1cm 1 at 492 nm. 

Texas Red hydrazide is soluble in DMF and may be dissolved as a concentrated stock solution in this solvent prior to the addition of a small aliquot to an aqueous reaction medium. The solid and all solutions made from it must be protected from light to avoid photodecomposition. Prepare the stock solution fresh immediately before use. A suggested protocol on the use of this fluorescent probe may be obtained by following the method outline for fluorescein-5-thiosemicarbazide in Section 1.1. Optimization may be necessary to achieve the best level of fluorescent modification (F/P ratio) for a particular application. 

---