Thermolysin fluorescence

A novel approach for determining metal-metal distances in proteins in solution was applied to thermolysin by Horrocks et al. (16), As mentioned earlier, thermolysin has four Ca2+ binding sites. Three of the four can be replaced by trivalent lanthanide ions. Tb3 + fluorescence is enhanced when Tb3+ is bound to the so-called 1 Ca2+ site of thermolysin. When Co2+ is added to the Zn2 + active site, the fluorescence of bound Tb3+ is quenched by... 

Another technique that uses the fluorescence properties of trivalent lanthanides is that of the detection of fluorescence emission decay induced by pulsed dye laser excitation. Horrocks and Sudnick (17) have applied this technique to the study of water molecules bound to metal ions in small complexes and proteins. In one study they found that the exponential decay of Tb3+ fluorescence is altered when H20 is replaced by D20 and that this change can be used to determine the number of coordinated water molecules on the metal ion. With thermolysin, bound Tb3+ had 1-2 water molecules in the first coordination shell. This number is consistent with the x-ray structure. 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

Figure 9.3.1 Ten amino acids were inserted at positions 1 and 2 in a pentapeptide. After acetylation the array of substrates was treated with the protease thermolysin. The liberated terminal amino groups were labeled with fluorescein isothiocyanate and the fluorescing spots detected with a scanning fluorescence microscope. The pentapeptide with amino acid 1 in both sites was obviously most easily hydrolyzed, followed by the ones with amino acid 1 in site 2 and amino acid 7 or 8 in site 1.

Cl shows an ESEM image of the particles. C2 shows fluorescence microscopy images of the particles loaded with a fluorescently labelled payload and C3 shows the removal of the payload from the particles after exposure to thermolysin. Source. Reproduced by permission ofThe Royal Society of Chemistry from (McDonald et al., 2009).)... 

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