Inhibition or initiation of a radical polymerization reaction by an ultraviolet-induced enzymatic process
Agfa-Gevaert Group, Мортсел, Flemish, BelgiumBiotechnology and Bioengineering (Impact Factor: 4.13). 03/1987; 29(4):403-13. DOI: 10.1002/bit.260290402
alpha-Chymotrypsin was modified to a light-controllable enzyme derivative by acylating active serine 195 residue with a cinnamoyl group or analogue. Upon UV irradiation the acylgroup could be isomerized, leading to release of the inhibiting group. Enzymatic activity could thus be regulated by means of UV light. A full 100% inhibition of the enzymatic activity could not be reached by the cinnamoyl derivative. Only posttreatment with diisopropylfluorophosphate yields a fully inactive enzyme derivative. The shelf-life of the inhibited enzyme was rather poor. Only freeze-dried samples could be used for several months without significant recovery of activity. Adapting the sensitivity of the system to visible light seems limited to the size of an enzyme's active site. Combination of the enzymatic system producing an inhibitor or an initiator with a polymerization reaction can result in a photographic process with a higher amplification factor.
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ABSTRACT: — Pyrenebutylmethylphosphonofiuoridate or pyrenepropylmethylphosphonofluoridate reacts with the serine 195 residue of α-chymotrypsin to yield a stoichiometric fluorescent derivative. The fluorescence decay of the modified enzyme was studied using a frequency-doubled, synchronously pumped picosecond rhodamine-6G laser excitation source with time-correlated single-photon-counting detection. Upon excitation above 330 nm, it could only be described adequately by a triple exponential decaying function. The exact reason for complexity of the emission has not yet been understood. Heterogeneous labeling seems irrelevant. The presence of different enzyme conformations on the single-photon-counting time-scale seems more plausible. The enzyme conformations where the pyrene label is situated close to the side groups of certain residues such as tryptophan and lysine show a much shorter fluorescence decay time resulting in non-exponential fluorescence behaviour. Dynamic fluorescence quenching experiments indicate that the pyrene label is located in a more polar region of the enzyme characterized by a negative charge. It seems that the pyrene group is too large to fit well in the hydrophobic pocket of the enzyme but is bent more towards the entrance of the cavity which is characterized by a higher polarity. Upon excitation at 296 nm, the excitation energy is transferred from the tryptophan chromophores towards the pyrene label.
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