Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution.

Paolo Ruzza,Giuliano Siligardi,Rohanah Hussain,Claudia Honisch

doi:10.3390/ijms222111325

Abstract

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Highlights

Circular dichroism (CD) spectroscopy is a valuable biophysical tool for studying the structure and conformation of biomolecules [1]
The estimation of the secondary structure elements (α-Helix, β-Strands, Turns and Unordered) of Bovine Serum Albumin fatty acid free (BSAff), human serum albumin (HSA) and hen egg white lysozyme (HEWL) shows a high content in α-helix, while IgG and Ubi possess a predominant β-sheet secondary structure and the CD spectrum of PLL is consistent with a left-handed extended helix of poly-proline type II [16,17]
Decrease in the intensity of both positive (192 nm) and negative (208 and 222 nm) bands was observed at increased number of scans indicating a progressive loss in the ordered structure from the estimation of the secondary structure content (SSE) using CONTNILL

Summary

Introduction

Circular dichroism (CD) spectroscopy is a valuable biophysical tool for studying the structure and conformation of biomolecules [1]. CD analysis in the far-UV region is widely used to evaluate the secondary structure of proteins/peptides in solution, as well as the influence of either ligands or environment (polarity, pH, ionic strength) on protein conformation and stability [2]. This analysis is often hampered by the buffer properties and the selection of the right buffer to use is a critical step. The highly collimated microbeam generated at B23 beamline of Diamond Light Source enables the measurements of CD spectra under a broad variety of conditions using: (i) capillaries with sub-milliliter volume of sample [3]; (ii) multiplate for high-throughput CD [4,5]; (iii) high pressure up to

Methods

Results

Conclusion