A Distinct Proof on Interplay between Trehalose and Guanidinium Chloride for the Stability of Stem Bromelain

Abstract

Guanidinium chloride (GdnHCl), a potential denaturant, is well-known to denature a number of proteins in vitro as well as in vivo studies. Its deleterious action on stem bromelain (BM) is quite prominent resulting decrease in protein structure and stability. The counteraction of this adverse effect of GdnHCl by the use of osmolytes is scarcely studied and the mechanism is still illusive and not exclusive. For the first time, to test elegant and simple counteraction hypothesis as a general mechanism we utilized fluorescence, circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering to study the counteraction of GdnHCl-induced denaturation of BM by the trehalose. It is revealed from the investigation of the results that trehalose is efficiently counteracting GdnHCl undesirable impacts on BM stability at molar ratio 1:1 of trehalose and GdnHCl. On the contrary, proteolytic activity of BM is increased only for the counteraction study of BM at very high concentrations of GdnHCl although still less than BM in buffer. The mutual exclusion of both trehalose and GdnHCl may stand for the counteraction of denaturation of BM resulting in a compact conformation with less solvent exposed surface area and increased secondary and tertiary structures. In addition, a decrease in BM-solvent interactions may also be contributing to some extent as there is little binding of trehalose replacing some water molecules and reducing binding of GdnHCl.

Coherent Experimental and Simulation Approach To Explore the Underlying Mechanism of Denaturation of Stem Bromelain in Osmolytes

“…By the accumulation of low molecular weight organic molecules, a diverse range of organisms from numerous phyla responded to the changes in intracellular water activity resulting from the hostile stresses such as dehydration, temperature variations, variable pH, freezing, high salinity, high concentrations of denaturants. − These molecules are termed as osmolytes or osmoprotectants. The methylamines and amino acids are major classes of naturally occurring osmolytes.…”

Activity of Bromelain with Cationic Surfactants and the Correlation with the Change of 1H NMR Signals

“…In the hydrophobic core of a folded protein Trp has high fluorescence intensity. In an unfolded protein, Trp is exposed to a polar environment and this results in a decrease in fluorescence (Rani and Pannuru, 2016; Rani and Venkatesu, 2015; Rani and Venkatesu, 2016). The combination of 1 H NMR data and Figure S5 suggested that bromelain and the surfactant together formed a more compact structure via hydrophobic interaction.…”

“…Studies on enhancing enzyme activity and stability have always been of great importance for both basic research and industrial applications (Arshad et al, 2014; Manzoor et al, 2016; Rani and Pannuru, 2016; Rani and Venkatesu, 2015; Rani and Venkatesu, 2016; Rathnavelu et al, 2016; Xue et al, 2019). The conformational stability of bromelain increases with increasing polyol size and the proteolytic activity of bromelain changes in the opposite order; among xylitol, erythritol, ethylene glycol, glycerol, and sorbitol, sorbitol is the weakest stabilizer for bromelain whereas it increases the activity of bromelain the most (Rani and Pannuru, 2016; Rani and Venkatesu, 2015).…”

“…GdnHCl and trehalose have opposite effects on bromelain activity; the former denatures bromelain while the latter stabilizes bromelain. However, the mutual exclusion of trehalose from GdnHCl causes the thermal stability of bromelain to increase only relative to that with GdnHCl, while the proteolytic activity is reduced (Rani and Venkatesu, 2015; Rani and Venkatesu, 2016). The stability and catalytic activity of bromelain can also be enhanced by chemical modification with anhydride groups (Xue et al, 2019).…”

Raman spectroscopic and quantum chemical investigation of the effects of trimethylamine N‐oxide on hydrated guanidinium and hydrogen‐bonded water networks

“…While guanidinium is structurally similar to urea having two amine groups, the exact mechanism by which guanidinium destabilizes proteins is largely unknown. [ 18–22 ] Moreover, there has been a great deal of controversy surrounding the molecular interactions between denaturants such as guanidinium and stabilizing osmolytes such as TMAO. One popular theory suggests that the stabilization of proteins is mediated through the direct interaction between the osmolyte and the protein, [ 12,23,24 ] which is commonly known as the “direct effect.” [ 25–30 ] Conversely, other studies have concentrated on the effects that the osmolyte has on hydrogen‐bonded water networks, which has been shown to affect the stability of the protein through the rearrangement of the solvation shell.…”