Journal Article

Aldehyde Production as a Calibrant of Ultrasonic Power Delivery During Protein Misfolding Cyclic Amplification

Drew, Simon C

2020

Overview

The protein misfolding cyclic amplification (PMCA) technique employs repeated cycles of incubation and sonication to amplify minute amounts of misfolded protein conformers. Spontaneous (de novo) prion formation and ultrasonic power level represent two potentially interrelated sources of variation that frustrate attempts to replicate results from different laboratories. We previously established that water splitting during PMCA provides a radical-rich environment leading to oxidative damage to substrate molecules as well as the polypropylene PCR tubes used for sample containment. Here it is shown that the cross-linking agent formaldehyde is generated from buffer ions that are attacked by hydroxyl radicals. In addition, free radical damage to protein, nucleic acid, lipid, and detergent molecules produces a substantial concentration of aldehydes (hundreds of micromolar). The measurement of aldehydes using the Hantzsch reaction provides a reliable and inexpensive method for measuring the power delivered to individual PMCA samples, and for calibrating the power output characteristics of an individual sonicator. The proposed method may also be used to better account for inter-assay and inter-laboratory variation in prion replication and de novo prion generation, the latter of which may correlate with aldehyde-induced cross-linking of substrate molecules.

• Publisher: Springer Nature B.V
• Subject: Aldehydes / Amplification / Containment / Crosslinking / Damage / Free radicals / Hydroxyl radicals / Laboratories / Lipids / Measurement methods / Nucleic acids / Polypropylene / Protein folding / Proteins / Sonication / Substrates / Tubes / Water splitting