ABSTRACT
The transmembrane domains of viral proteins are highly conserved and crucial to normal viral function. Oligomeric transmembrane domains present novel opportunities for drug development, as their disruption can prevent the assembly of the virus. The Reichart lab is particularly interested in developing retro-inverso peptide inhibitors. Retro-inverso peptides are peptides using D-amino acids mirroring a region of target protein, which allows the peptide to inhibit viral assembly, but they are also significantly less likely to be catabolized by natural metabolic or immunologic processes. The efficacy of these inhibitors is governed largely by the extent to which they mirror the target protein, making highly conserved regions, such as transmembrane domains, ideal target regions for these inhibitors. The primary technique in the literature for the investigation of oligomerization states uses fluorescence spectroscopy. We are now working on developing a novel alternative system to evaluate protein oligomerization using spin-labeled peptides that are directly incorporated into the peptide sequence. Direct incorporation of the spin-label into the peptide sequence is a more powerful technique than the standard procedures used in the literature. In particular, the ability to incorporate spin labels in various positions within the protein can give novel insights into the relative depth of the protein within a membrane, which is very difficult to study using other techniques and not possible using the fluorescence technique. The transmembrane domains of proteins with known and well-characterized monomer and trimer standard oligomerization states were synthesized using an Fmoc Solid- Phase Peptide Synthesis (SPPS) procedure incorporating an Fmoc-2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, (Fmoc-TOAC) instead of an alanine. Direct incorporation of stable N-oxide spin labels, which can be contrasted to labeling cysteine residues after the protein synthesis, has been used for the investigation of the secondary structure of proteins for decades, but the application of this spin labeling technique to study the oligomerization states of transmembrane domains of proteins is an understudied application. The products of SPPS were analyzed using a Liquid Chromatography Mass Spectroscopy instrument and purified using High Performance Liquid Chromatography. The spin-label was then deprotected and evaluated using Electron Spin Resonance (ESR) Spectroscopy. There are two primary future directions following this research project: first, the generation of viral proteins with spin labels incorporated in different positions to determine the relative depth of each position within the membrane;second, the incorporation of spin labels into SARS-CoV- 2 proteins to develop a model for in vitro evaluation of retro-inverso peptide assembly inhibitors. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Ascorbic acid has recently been extensively used due to its role in the management of COVID-19 infections by stimulating the immune system and triggering phagocytosis of the corona virus. The currently used spectrofluorometric methods for determination of ascorbic acid require using derivatizing agents or fluorescent probes and suffer from a number of limitations, including slow reaction rates, low yield, limited sensitivity, long reaction times and high temperatures. In this work, we present a highly sensitive spectrofluorometric method for determination of ascorbic acid by switching-on the fluorescence of salicylate in presence of iron (III) due to a reduction of the cation to iron (II). The addition of ascorbic acid resulted in a corresponding enhancement in the fluorescence intensity of iron (III)-salicylate complex at emission wavelength = 411 nm. The method was found linear in the range of 1-8 µg/mL with a correlation coefficient of 0.9997. The limits of detection and quantitation were 0.035 µg/mL and 0.106 µg/mL, respectively. The developed method was applied for the determination of ascorbic acid in the commercially available dosage form; Ruta C60® tablets. The obtained results were compared with those obtained by a reported liquid chromatographic method at 95% confidence interval, no statistically significant differences were found between the developed and the reported methods. Yet, the developed spectrofluorometric method was found markedly greener than the reference method, based on the analytical Eco-scale and the green analytical procedure index. This work presents a simple, rapid and sensitive method that can possibly be applied for determination of ascorbic acid in pharmaceuticals, biological fluids and food samples.