Fluorescence lifetime albumin

P. Suci and V. Hlady, Fluorescence lifetime components of Texas Red-labeled bovine serum albumin Comparison of bulk and adsorbed states, Colloids Surf. 51, 89-104 (1990). 

Quin-2 binds to human serum albumin and forms a complex with the protein. Thus, we are in presence of a static quenching. In this case, one should not observe any variation in the fluorescence lifetime of the Trp residues of the protein. However, this is not the case (Table 3.3) and the decrease in the fluorescence lifetime is in the same order of that of the intensity. This is explained by the presence of an energy transfer from the Trp residues to Quin-2. 

Utilizing time resolved internal reflection spectroscopic technique (Fig. 6), we were able to isolate the tryptophan intrinsic fluorescence and observe its = 20 ns fluorescence lifetime for albumin in bulk and in the surface microenvironment of a hydrophilic quartz material. The pH dependence of bulk albumin fluorescence lifetime served to "calibrate albumin in terms of native ( 7 ns time constant) protein at pH 7.2 and unfolded (c 4 ns) protein at the isoelectric pH 3.8. The fluorescence lifetime data (Tables I/II) supported the hypothesis that the adsorbed albumin exists in two forms on a hydrophilic quartz surface, each with a possibly different structure (] ). A loosely held "layer," consisting of microaggregates, native and partially unfolded albumin molecules with... 

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.

In considered model (la), the fluorescence saturation is caused by a finite lifetime r and by intercombination conversion. In model (lb), due to the finite fluorescence lifetime and due to the saturation of the energy transfer chaimels. Let us note that the model (lb) could be also supplemented with the intersystem crossing mechanisms, but paehmtnaiy expieiiments showed that at the given parameters of the laser radiation the process for albumins and mRFPl is small compared to the mechanisms under study and contributes little to fluorescence saturation. Therefore, this mechanism has been excluded to increase the stability of the inverse problem solution (details and mathematical basement of inverse problem solution of nonlinear laser fluorimetry can be found elsewhere (Boychuk at al., 2000). For the same reason the induced processes from the excited states (two-photon absorption or photoizomerization, etc) have been excluded. 

Titration of constant concentration of human serum albumin with Quin-2 induces a concomitant decrease of both the fluorescence intensity and lifetime of the Trp residues of the protein (Table 3.3). 

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