A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed.
Three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4N3-IAA, 5-N3-IAA, and 6-NA-IAA) have been synthesized for use as fluorescent photoaffinity labeling agents. The pKa values of these compounds (4-N3-IAA, 4.67; 5-Ns-IAA, 4.65; 6-N3-IAA, 4.66; all ± 0.04) are experimentally indisfinguishable from the pK. of 3-indoleacetic acid (IAA, 4.69 ± 0.04). The auxin activity of these IAA derivatives has been determined in several systems. In soybean, pea, and corn straight growth assays, all three analogs induce growth comparable to that caused by IAA. In the tobacco pith assay, anl three analogs elicit a maximum increase in fresh weight at least 40 to 50% of that caused by IAA. Optimal growth is attained in the tobacco pith assay at slightly higher concentrations of 4Ns-IAA and 6-N3-IAA (30 micromolar) than required for IAA (10 micromolar); however, maximal growth is achieved at a slightly lower concentration of 5-N3-IAA (3 micromolar). The NA-IAAs, like IAA, are transported basipetally through tobacco pith tissue.Auxins, the longest known class of plant hormones, modulate a wide variety of cell functions, the most significant being cell elongation (21,31,39,42,54,55). Despite extensive investigation, the mechanism of auxin action is still unknown, although it is now generally recognized that the response to auxins occurs in two phases (58,60,61). Within the past decade, reversible binding between auxins and several cell components has been demonstrated, establishing tentative locations of auxin receptors within the cell (17, 19, 23, 27, 43, 45, 50-52, 59, 61-66) (41).The decline in the rate of appearance of reports on auxin binding in the past 2 years compared to the rate during the 6 years immediately following the initial demonstration of auxin binding (23) and recent attempts to predict the properties of the auxin receptor from empirical structure-activity correlations (29,33,49) signal the need for a fresh approach to the problem of isolating an auxin receptor.To this end, we have synthesized three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4-N3-IAA, 5-N3-IAA, and 6-N3-IAA)7 for use as fluorescent photoaffinity labeling agents. The technique of photoaffinity labeling, which has been used to study animal hormones and to characterize the active sites of enzymes, has been thoroughly reviewed (6, 14-16, 30, 32
The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N3IAA), and of the photoproducts of 5-N3IAAto the naphthalene-l-acetic acid (NAA) binding sites of Zea mays L. WF9 x BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4-and 5-N3IAA bind to these sites with affinities similar to that of IAA, while 6-N3IAA and the photoproducts of 5-N3IAA bind less tightly. This 3School of Chemical Sciences. 4Abbreviations: NAA, naphthalene-l-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; NAA, naphthalene-1-acetic acid; N3IAA, azidoindole-3-acetic acid; 4-N3IAA, 4-azidoindole-3-acetic acid; 5-N3IAA, 5-azidoindole-3-acetic acid; 6-N3IAA, 6-azidoindole-3-acetic acid; RAC, ratio of association constants.that avoids the requirement that conditions for equilibrium binding be maintained while searching among the membrane proteins for specific auxin-binding sites. Once binding proteins are labeled, they can be isolated using standard techniques without the need to maintain equilibrium binding conditions. Therefore, the advantage of photoaffinity labeling is that binding proteins can still be isolated after they have lost the capacity for binding. Photoaffinity analogs bind reversibly to a site in the dark but can be caused to bind irreversibly by irradiation with light. Light induces the formation of reactive intermediates that attack nearby groups to form covalent bonds. The effectiveness of a photoaffinity agent, therefore, depends upon its affinity for the binding site in the dark and upon the half-lives of these photogenerated intermediates (4,24). The half-life of the ligandbinding site complex must be considerably longer than the halflives of the intermediates to achieve specific labeling.The only previous attempt to label the auxin site by affinity labeling was by Venis (26). He tested the usefulness of the diazonium salts of two auxin analogs, 2-chloro-4-aminophenoxyacetic acid and 2,5-dichloro-3-aminobenzoic acid, and reached tentative conclusions about the involvement of several amino acids in the auxin active site. The potential photoaffinity labeling agents 4-azido-2-chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid were found to have only moderate auxin activity (15), unlike the azidoindole-3-acetic acids here described. Spring and Hager (25) have recently demonstrated that sesquiterpene lactones specifically label important growth sites only when an auxin is also bound. The experiments provide an example of a nonequilibrium binding technique useful in auxin receptor research and are important because the results support a molecular model for auxin binding. The authors propose that auxin induces the receptor to undergo a conformational change, important for cell growth. Most significantly, the technique offers an independent assay for identifying auxin receptors and could be useful in conjunction with an independent approach such as we propose in this report.We ...
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