HPLC Assessment of synergy between non-reducing sugars and intrinsically disordered proteins in protecting fragile biological materials from desiccation.
Abstract
Society takes advantage of the wide availability of refrigeration to maintain food, medical supplies (vaccines, plasma), chemicals, and whole cells in a useful state. The cold-/cool-chain (CC; as it has become known) ensures that valuable, fragile biological materials (FBM) are stored under conditions that maximize FBM utility. The absolute requirement for uninterrupted CC maintenance to safeguard FBM function underlies the vulnerability of the CC to electrical disruption. Power outage frequently occurs during natural disasters and armed conflict while remote areas remain without access to electricity entirely. Drying FBM while maintaining its serviceability (usually defined as activity or even viability upon rehydration) offers numerous advantages relative to the CC which is expensive, difficult to extend to regions without it, subject to disruption, centrally distributed, and conspicuous by its nature (heavy, bulky, noisy refrigerators/generators and equipment heat signatures at a central locale). The focus of my research is to understand how certain organisms can, at some stage of their life cycle, withstand almost complete loss of water without dying. While in the desiccated state these organisms display extreme resistance to abiotic perturbation and aging. Generally, the attributes cells acquire leading to anhydrobiosis (life with little water) include the accumulation of specific, non-reducing oligosaccharides (trehalose, and sucrose:raffinose admixtures) and some form of intrinsically disordered protein (in plants and some animals, LATE EMBRYOGENESIS ABUNDANT PROTEINs, LEAPs). These macromolecules are thought to: 1) replace water around otherwise desiccation sensitive cellular constituents; 2) vitrify the cellular interior, embedding cellular contents in a glass that severely restricts movement and represses metabolism and; 3) shield sensitive cellular components from reactive oxygen species and denaturation. Our current scientific objective is a step-wise investigation of how the hallmark anhydrobiotic molecules (oligosaccharides and LEAPs) perform in synergy to enhance the stability of FBMs to desiccation. The first step is to assess the labile enzyme LACTATE DEHYDROGENASE (LDH) and large, unilamellar vesicles (LUVs) for their capacity to withstand desiccation in the presence of various admixtures of oligosaccharides and intrinsically disordered LEAPs. To do this we will assess enzyme activity (LDH) and vesicle integrity (vesicle size and release of Calcein fluorescent dye). LDH activity will be monitored after freezing in liquid nitrogen as well as after desiccation for various time periods. The loss of Calcein from pre-loaded LUVs after desiccation will be followed using a SpectraMax M2 fluorescence microplate reader (Molecular Devices Corp. Sunnyvale, CA) at an excitation wavelength of 444 nm and an emission wavelength of 555 nm, providing a measure of vesicle integrity. Protective oligosaccharide production and the capacity to quantitatively recover these oligosaccharides from freshly harvested versus freeze-dried leaves, as well as assessments of the protective sugar amounts provided to the LDH activity- and LUV integrity-assays, will use the High Pressure Liquid Chromatograph that was the subject of this award. Two undergraduate researchers will be recruited from the University of Kentucky (UK) Medical and Agricultural Biotechnology program to assist in this project. They will help forward our rudimentary understanding of how two groups of molecules, polyhydric, non-reducing sugars and intrinsically disordered, hydrophilic LEAPs may protect labile macromolecules from desiccation-induced damage. The relative effectiveness of the protective sugar trehalose versus 3:1 sucrose:raffinose in defined concentrations in affording protection to FBM will be ascertained. The synergy in the protective influence of specific sugars and LEAPs, in defined ratios and amounts, will be revealed.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 20, 2019
- Source ID
- W911NF1910222
Entities
People
- A Downie
Organizations
- Army Contracting Command
- United States Army
- University of Kentucky