Protein excitation

E.g. tryptophane residues of proteins excite at 290-295 mn but they emit photons somewhere between 310 and 350 mn. The missing energy is deposited in the tryptophane molecular enviromuent in the form of vibrational states. While the excitation process is complete in pico-seconds, the relaxation back to the initial state may take nano-seconds. While this period may appear very short, it is actually an extremely relevant time scale for proteins. Due to the inherent thermal energy, proteins move in their (aqueous) solution, they display both translational and rotational diffusion, and for both of these the characteristic time scale is nano-seconds for normal proteins. Thus we may excite the protein at time 0 and recollect some photons some nano seconds later. With the invention of lasers, as well as of very fast detectors, it is completely feasible to follow the protein relax back to its ground state with sub-nano second resolution. The relaxation process may be a simple exponential decay, although tryptophane of reasons we will not dwell on here display a multi-exponential decay. 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

Protein Excited-State and Ground-State Energy Storage).151... 

Figure 18 Low-temperature (77 K) RR spectra in the 200-500 cm region of indicated blue Cu proteins excited at 568.2 (stellacyanin, NiR) and 647.1 run (azurin). Asterisks denote ice band...

A serum or urine sample contains many compounds that fluoresce. Thus the sample matrix is a potential source of unwanted background fluorescence and must be examined when new methods are developed. The most serious contributors to unwanted fluorescence are proteins and bilirubin. However, because protein excitation maxima are in the spectral region of 260 to 290 nm, their contribution to overall background fluorescence is minor when excitation occurs above 300 nm. 

The fluorescence maxima of DMOL and MMOL shift to the short-wave region upon their binding the enzyme 20 nm for DMOL and 100 nm for MMOL, and the fluorescence intensity significantly increases (Fig. 3). The observed changes in the fluorescence emission spectra of DMOL can be explained by the increase in hydrophobicity of microenvironment of the emitter on its binding with the protein. Excitation spectrum of MMOL-luciferase complex (Xem=450 nm) corresponds to the absorbance spectra of MMOL-monoanion. In buffer solution we did not seen the fluorescence spectra of this form due to the fact that in water solution electronically excited MMOL-monoanion exists in form of phenolate-ion only (it is in dianion form). The luciferase microenvironment stabilizes MMOL-monoanion by protecting its phenolic group from dissociation. It shows that in vicinity of MMOL bound to... 

Also, the Phe-64 Leu and Ser-65 Thr mutant shows also higher fluorescence parameters comparative to the wild green fluorescent protein. Excitation at 458 nm yields a fluorescence spectrum with two peaks at 512 and 530 nm The fluorescence properties of this enhanced green fluorescent protein (EGFP) were found similar to the recombinant glutathione S-transferase-EGFP (GST-EGFP) protein, expressed in Escherichia coli (Cinelli et al. 2004). 

Alpert, B. and Lopez-Delgado, R, 1976, Fluorescence lifetimes of haem proteins excited Into the tryptophan absorption band with synchrotron radiation. Nature, 263, 445 - 446. 

Figure 3. Model for the structure of photosystem I, showing the distribution of chlorophyll a and b between the component chlorophyll-proteins. Excitation energy transfer may occur in a linear sequence from LHCII (under state 2) to CPI, The fluorescence emission peaks for the individual chlorophyll-proteins are also shown, although are usually shifted in vivo, when they are associated with other chlorophyll-proteins.

When performing denaturant titrations using fluorescence, either Protocol 7 or 2 can be followed. When monitoring the fluorescence of tryptophan residues in a protein, excitation and emission wavelengths of 290 nm and 350 nm, respectively, are typical. It is important to also measure the fluorescence signal from the denaturant solution and to subtract this blank signal from the fluorescence signal from the protein solutions. 

---