Protein quenching correction

Figure 3. Quenching of the fluorescence spectrum of MeUmbG2 by CBH II from Trichoderma reesei (5). Curve A represents the MeUmbGj (2 /iM) spectrum in the absence of CBH II. Curves B, C and D show the spectra after the addition of several aliquots of 137.7 /iM CBH II. Spectrum D changes to E when solid cellobiose ( 2 mg) is added. When correction is made for dilution, spectrum E is equivalent to spectrum A. Curves F said G represent buffer and protein blanks, respectively. Spectra were measured at pH 5.0 and 6.6°C.

Details of several years of UNF research exploiting rapid reaction, rapid quench and stopped flow kinetics are in the scientific literature and would be inappropriate here. The upshot was a scheme which evolved over the period and, with later refinements, is now considered to be correct in all its major features. In brief, the obligatory evolution of dihydrogen is believed to arise from the formation of a metal-dihydride complex within the reduced MoFe-protein, a two-step process requiring the transfer of two electrons successively from the MgATP-activated Fe-protein coupled with two protonation steps and the hydrolyses of four molecules of ATP to ADP N2 is bound by displacement of two... 

Despite the fact that the enhancement of RME fluorescence associated with RME binding to CRBP might be exploited to perform titrations of CRBP with the retinoid, It is more convenient to monitor the interaction between RME and CRBP by following the quenching of intrinsic protein fluorescence. In particular, the titration method based on the enhancement of RME fluorescence is remarkably less sensitive than the method based on the quenching of protein fluorescence In addition, a correction for the contribution of the fluorescence of free retinoid present m the medium should be made for each point of a titration based on the enhancement of RME fluorescence... 

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