Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(hydroxymethyl)aminomethane

Abstract

Fast photochemical oxidation of proteins (FPOP) is a powerful covalent labeling tool that uses hydroxyl radicals generated by laser flash photolysis of hydrogen peroxide to footprint protein surfaces. Because radical production varies with many experimental parameters, hydroxyl radical dosimeters have been introduced to track the effective radical dosage experienced by the protein analyte. FPOP experiments performed using adenine optical radical dosimetry containing protein in Tris buffer demonstrated unusual dosimetry behavior. We have investigated the behavior of Tris under oxidative conditions in detail. We find that Tris can act as a novel gain-of-signal optical hydroxyl radical dosimeter in FPOP experiments. This new dosimeter is also amenable to inline real-time monitoring, thereby allowing real-time adjustments to compensate for differences in samples for their quenching ability.

Figure 1.

Absorbance of Tris at 265 nm as measured by NanoDrop after exposure to four sample conditions. Absorbance increases significantly only upon reaction with hydroxyl radicals generated by flash photolysis of hydrogen peroxide.

Figure 2.

Tris absorbance at 265 nm correlates positively with average oxidation of GluB peptide.

Figure 3.

A) Tris absorbance change for myoglobin samples without MES scavenger, with 10 mM MES scavenger, and compensated conditions with 10 mM MES scavenger and increased laser fluence to obtain a ΔAbs₂₆₅ ≈ 4.97. B) (Blue) Peptide oxidation for myoglobin peptides in the absence of MES; (Orange) Peptide oxidation for myoglobin peptides in the presence of 10 mM MES; (Grey) Peptide oxidation for myoglobin in the presence of 10 mM MES under compensating laser fluence conditions, using Tris as a doseimeter for radical compensation. No statistically significant differences were detected in peptide oxidation between no MES samples and with MES-containing samples compensated using Tris dosimetry.