Inert Gas Deactivates Protein Activity by Aggregation

Abstract

Biologically inert gases play important roles in the biological functionality of proteins. However, researchers lack a full understanding of the effects of these gases since they are very chemically stable only weakly absorbed by biological tissues. By combining X-ray fluorescence, particle sizing and molecular dynamics (MD) simulations, this work shows that the aggregation of these inert gases near the hydrophobic active cavity of pepsin should lead to protein deactivation. Micro X-ray fluorescence spectra show that a pepsin solution can contain a high concentration of Xe or Kr after gassing, and that the gas concentrations decrease quickly with degassing time. Biological activity experiments indicate a reversible deactivation of the protein during this gassing and degassing. Meanwhile, the nanoparticle size measurements reveal a higher number of "nanoparticles" in gas-containing pepsin solution, also supporting the possible interaction between inert gases and the protein. Further, MD simulations indicate that gas molecules can aggregate into a tiny bubble shape near the hydrophobic active cavity of pepsin, suggesting a mechanism for reducing their biological function.

Figure 1

Outline and representative results in this study. The upper row shows micro X-ray fluorescence measurements in the pepsin system during the saturated gassed and degassed states, using Xe as an example. From left to right, the black and blue curves refer to the absorption of the pepsin system (0.5 mg/ml) without Xe and after gassing with Xe, respectively. The right curve shows the absorption of Xe in the pepsin solution after degassing for about 2 hours. The middle row shows the activity of pepsin measured in the saturated gas and degassed states together with the control system. The bottom row displays the MD simulation results of the backbone root-mean-square deviation (RMSD) and the conformations of pepsin with Kr, Xe, and N₂ aggregations/bubbles and after degassing. In all results the black colour shows the data of the initial degassed system while the light blue, blue, and red colours represent the data of Kr, Xe, and N₂.

Figure 2

Micro X-ray fluorescence absorption near (a) Xe L-edge and (b) Kr K-edge in a pepsin solution, HCl solution without pepsin, and degassed water.

Figure 3

Micro X-ray fluorescence absorption of (a) Xe and (b) Kr with degassing time in a solution of 0.1 mg/ml pepsin.

Figure 4

The distribution of “particles” measured in a pepsin solution with and without Xe as well as Xe saturated HCl solution. The distribution of nanoparticles differs from the system without Xe. From the curves, the number of “particles”, especially nanometre-scale particles, increases after injecting Xe in the pepsin solution.

Figure 5

(a) MD simulations of the degassing process over 80 ns. (b) Enlargement showing the range from 60 ns to 80 ns.