Category: Chemical Biology

1039-E - Synergy between 275 nm and 365 nm UV LEDs for inactivation of RNaseA

Wednesday, February 7, 2018
11:30 AM - 12:30 PM

We have recently determined that a high irradiance 275 nm UVLED source inactivates RNaseA at lower doses (joules/cm2) than lower irradiance source (1). Inactivation using 275 nm disrupts the disulfide bonds (2) but does not directly affect the histidine or lysine amino acids known to be located in the enzyme’s active site. In this study, 365 nm UV LED (a wavelength that approximates the bond dissociation energy of 331 kJ/mol for CH3-NH2 group present in the lysine amino acid side chain) was investigated for its effect on RNaseA enzyme activity. RNaseA contaminated dry surfaces (1 µl 0.02 U/ml RNaseA) were exposed to UVLEDs from a distance of 25 mm. The RNaseA sample was recovered from each surface in RNase free water and the suspension fluorometrically assayed for RNase activity (RNaseAlert IDT). RNaseA samples were exposed to 275 nm alone (high irradiance - 38.5 mW/cm2 and low irradiance - 18.7 mW/cm2 at the target), to 365 nm alone (1.35 W/cm2 at the target), and to 275 nm and 365 nm concurrently (18.7 mW/cm2 and 1.35 W/cm2, respectively). The 275 nm UV LEDs inactivated RNaseA at doses of 6.9 J/cm2 for the 38.5 mW/cm2 source and 22.5 J/cm2 for the 18.7 mW/cm2 source. Exposure to 365 nm alone at doses up to 2400 J/cm2 had no effect on enzyme activity - tracking the positive control. When concurrently exposed to the low irradiance 275nm and the 365 nm sources, RNaseA was inactivated at a dose of 5.6 J/cm2 (275 nm source) and 406 J/cm2 (365 nm source). This result shows a synergy between the effects of 275 nm and 365 nm wavelengths for RNaseA inactivation, suggesting that the use of multiple targeted wavelengths is a viable path for control and rapid inactivation of proteins and enzymes.
1) Pasquantonio J, Eliason G, and Thompson T. Rapid inactivation of RNaseA by high irradiance UV LEDs (Submitted).
2) Neves-Petersen MT, Gryczynski Z, Lakowicz J, Fojan P, Pedersen S, Peterson E, and Bjørn Petersen S. 2002. High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue. Protein Sci., 11(3) pp588-600.

Theresa L. Thompson

Applications Scientist/Engineer
Phoseon Technology
Hillsboro, OR

Theresa Thompson holds a Ph.D. in Molecular Biology from the University of Southern California and is curently an Applications Scientist at Phoseon Technology. She comopleted postdoctoral studies at Children's Hospital Los Angeles and has since worked in industry as a Senior Scientist at Oligos Etc working in DNA synthesis, analysis and oligonucleotide design, as VP Research at Dimera Inc working in cardiovascular drug development, and as Chief Scientist at Chimerochem (a small start-up) working on monomer chemistry.