Abstract Certain marine organisms produce calcium-ac- tivated photoproteins that allow them to emit light for a variety of purposes, such as defense, feeding, breeding, etc. Even though there are many bioluminescent organ- isms in nature, only a few photoproteins have been iso- lated and characterized. The mechanism of emission of light in the blue region is the result of an internal chemi- cal reaction. Because there is no need for excitation through external irradiation for the emission of biolumi- nescence, the signal produced has virtually no back- ground. This allows for the detection of the proteins at ex- tremely low levels, making these photoproteins attractive labels for analytical applications. In that regard, the use of certain photoproteins, namely, aequorin, obelin, and the green fluorescent protein as labels in the design and de- velopment of binding assays for biomolecules has been reviewed. In addition, a related fluorescent photoprotein, the green fluorescent protein (GFP), has been recently employed in bioanalysis. The use of GFP in binding as- says is also discussed in this review. Introduction Bioluminescence can be defined as the production of vis- ible light via chemical reaction that occurs within a living organism. This important natural phenomenon is em- ployed by many creatures for purposes of defense, mat- ing, concealment, and the recycling of nutrients [1]. There is a variety of luminescent organisms in nature that in- cludes worms, insects, bacteria, crustaceans, fireflies, fungi, and fish. Within these organisms, there are approx- imately 30 different bioluminescence systems that have evolved independently and which are still in existence [2]. Each luminescent system involves an exergonic reaction of molecular oxygen with the protein and substrate to pro- duce light within the visible region. The enzymes in- volved in the bioluminescence reaction are oxygenases, which are generically referred to as luciferases [3]. The substrates for these enzymes are commonly termed lu- ciferines, which means “light bearers” [2]. Luciferases commonly employ molecular oxygen to oxidize the bound luciferins, producing molecules in an excited state. luciferin + O 2 (or H 2 O 2 ) luciferase oxyluciferin* oxyluciferin* oxyluciferin + hν The substrates for different bioluminescent reactions vary substantially in chemical structure, and essentially, they are chemically unrelated. Examples of various biolumi- nescent organisms and their corresponding substrates are shown in Table 1 [2]. The production of light also varies according to the particular bioluminescent organism. Some organisms are capable of emitting light continuously, while others emit light as a rapid flash lasting less than 10 s. In these cases, the enzymatic reaction is turned on rapidly, requiring the appropriate reagent to be sequestered and mobilized quickly. Triggering agents for the bioluminescent flash- type light emission systems vary, and some examples in- clude a rapid pH change within an organelle present in the organism, the presence of oxygen, or the binding to cal- cium ions [3]. A particular class of proteins known as photoproteins emit a rapid flash of light upon binding to calcium ions. The use of these proteins in bioanalysis, along with their associated green fluorescent proteins (GFP) [4], will be the focus of this review. Calcium-dependent photoproteins are used to produce light by the bioluminescent organisms of the phyla Proto- zoa, Cnidaria, and Ctenophora. Two classes of Cnidaria, Hydrozoa and Anthozoa have been studied in depth [5]. One hydrozoan in particular, the jellyfish Aequorea victo- ria (Fig. 1), was the first organism from which a photo- protein (aequorin) was isolated. This was achieved by Shimomura’s group in 1962. A. victoria is 4 inches in dia- meter and shaped like a hemispherical umbrella (see Fig. 1). Jennifer C. Lewis · Sylvia Daunert Photoproteins as luminescent labels in binding assays Fresenius J Anal Chem (2000) 366 : 760–768 © Springer-Verlag 2000 Received: 7 November 1999 / Revised: 25 January 2000 / Accepted: 26 January 2000 REVIEW J. C. Lewis · S. Daunert () Departments of Chemistry and Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40506-0055, USA e-mail: daunert@pop.uky.edu