Fluorescence efficiencies, amino acids

Reymond and coworkers designed a new enzyme assay utilizing an M-IDA." The fluorescence of a quinacridone indicator 30 is quenched by formation of a macrocycUc chelate M-30 (M = Cu or Ni " ). Because of a large 17-membered ring in the chelate, this complex is relatively unstable. As a result, other weak metal chelators, such as amino acids, can efficiently compete with 30 for metal com-plexation. After the metal is released from the indicator, the fluorescence is restored. Two enzymatic reactions that produce amino acids from noncoordinating substrates were successfully monitored. Figure 16 shows the fluorescence response of 30 in the presence of metal ions during enzymatic reactions. Another indicator, calcein (31), which is commercially available, was also employed in a similar... 

Taki, M., Hohsaka, T, Murakami, H., Taira, K., and Sisido, M., A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe, FEES Lett., 507,1, 35, 2001. 

A number of drawbacks in the application of the 0PA/2-ME reagent system include the instability of the fluorescent isoindole derivative (5-7) the use of the noisome reagent 2-mercaptoethanol the low and solvent-dependent fluorescence efficiencies (8,9) of the isoindole and—perhaps the most limiting—the effective restriction of the OPA assay to primary aliphatic amines and to amino acids. 

Perhaps most encouraging in these discoveries was the observation that NDA/CN worked equally well for derivatization of dipeptides and higher homologues of the primary amino acid series. Again, a stable, fluorescent, isolatable derivative was obtained. One of the most important initial findings was the high fluorescence efficiency of the CBI adduct (12). Tables 1 and 2 list the efficiencies for a representative group of mono-, di-, and tripeptides and a limited comparison of the CBI efficiencies with the more traditional OPA (8) and dansyl (9) derivatives, respectively. 

Recently, chloromethylated benzocoumarin 11c, hydroxylmethylated benzocou-marin 12, and chloromethylated coumarin 13 were used in the efficient preparation of several fluorescent ester conjugates of /V-benzyloxycarbonyl-neurotransmitter amino acids, such as p-alanine, tyrosine, 3,4-dihydroxyphenylalanine (DOPA), glutamic acid, and y-aminobutyric acid (GABA) [39, 40],... 

Dialkylamino phthalic acids resulting from the chemiluminescence reaction of 4-dialkylamino-phthalic hydrazides cannot easily form a tris anion because deprotonation of the amino group is impossible. They should therefore not exhibit such a strong decrease in fluorescence efficiency at higher pH values. This can actually be concluded from the pH dependence of the chemiluminescence of the 4-dialkylamino-phthalic hydrazides and related compounds 97>. 

Because of the importance of validating the moist-heat treatments and related processes, it is necessary to develop a BI that can efficiently assess the success of the treatment. The use of a fluorescent marker designed for a quick and reliable assay, detectable by microscopy, spectrofluorometry, or a handheld UV lamp, is examined herein. GFPuv provides the basis for its potential utility as a fluorescent BI, to monitor moist-heat treatments (T < 100°C). GFPuv is a compact, globular acidic protein (p/ 4.6-5.4) with one fluorophore consisting of a cyclic tripeptide in the primary protein sequence, a chain of 238 amino acids. It has been shown to be resistant to heat (T > 70°C) and alkaline pH (between 5.5 and 12.0 optimum = 8.0). 

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