Protease fluorescence lifetime

Doering K, Meder G, Hinnenberger M, Woelcke J, Mayr LM, Hassiepen U (2009) A fluorescence lifetime-based assay for protease inhibitor profiling on human kallikrein 7. J Biomol Screen 14 1-9... 

The same publication indicated that fluorescence lifetimes of compounds from the compound collection of Pfizer classified as problematic due to their autofluorescence characteristics resulted in false positive results in many FI-based assays. For most compounds, the fluorescence lifetimes were below 1 ns. Thus, FLT measurements with a reporter fluorophore displaying a lifetime significantly longer than 1 ns are suitable for application in protease assays for compound testing. However, for applications under initial velocity conditions with a substrate turnover below 20%, fluorophores with lifetimes of a few nanoseconds are still critical because the dynamic range of the assay is then too low with lifetimes below 1 ns. 

Nevertheless, let us discuss now the HIV -1 protease fluorescence in terms of rotamers model. The purpose of this discussion is to find out how far we can go in the application of this model. Therefore, let us consider the rotamers model as correct and thus the two fluorescence lifetimes (0.5 and 3.1 ns) of free Trp in solution originates from two rotamers. If this model is correct in proteins also and thus in HIV-1 protease, we have two tryptophans with four lifetimes, two short (close to 0.5 ns) and two long (close to 3.1 ns). Thus, we can attribute to each tryptophan, long and short fluorescence lifetimes. In other terms, the presence of the protein matrix around Trp residues does not play any fundamental role in the fluorescence lifetimes of the Trp residues. In this case, the protein backbone has no effect or non significant effect on the fluorescence lifetime of HIV-1 protease. In simple words, the fluorescence lifetime of tryptophan would be in dependent of the tertiary structure of HIV-1 protease. 

Figure 5. Hydrolysis of fluorogenic substrate 1 by HTV protease at 37 C as monitored by steady state fluorescence spectroscopy (Xex+340, A m=490). The reaction was carried out with 10 pM substrate at pH 4.7 in a buffer solution containing 0.1 M NaOAc, 1.0 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% DMSO and 1 mg/mL bovine serum albumin. The arrow indicates the point of addition of HTV protease to a final concentration of 35 nM. Product analysis was carried out by HPLC, mass spectrum and fluorescence lifetime. Inset The initial phase of the hychx)lysis reaction used for rate determinations.

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

Protease assays based on the FLT of PT14 are especially attractive due to the long lifetime of the dye of 14 ns that allows the discrimination of the lifetimes of short-lived fluorescent compounds from that of PT14. Thus, the rate of false-positive and -negative results can be reduced. The major... 

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