Terahertz Spectroscopic Study of Ion Effects on Protein Hydration Toshiaki Hattori, Katsuyoshi Aoki, and Kentaro Shiraki Institute of Applied Physics, University of Tsukuba Email: hattori@bk.tsukuba.ac.jp Abstract—Effects of salts on the dynamical properties of hydration water of protein in aqueous solutions are studied using high-precision terahertz spectroscopy. Kosmotropic anions in the well-known Hofmeister series are found to decrease hydration water, and chaotropic anions increase it. This result is consistent with a theoretical model which describes the ion effects on hydration of proteins in terms of the hydrogen-bonding network around the protein. I. I NTRODUCTION AND BACKGROUND C ONTROLING solubility, folding, and stability of pro- teins has been an important subject in protein engineer- ing, pharmaceutics, and food industry. It has been known that the ability of anions to precipitate proteins and also anion effect on the stability of proteins are ordered in the Hofmeister series: SO 2- 4 > H 2 PO 4 - > Cl - > NO - 3 > SCN - . The left-side anions are called kosmotropes (structure makers), and the right-side anions chaotropes (structure breakers). The microscopic mechanism underlying the Hofmeister series has been discussed, and hydration of protein molecules is expected to play the critical role although clear experimental demonstra- tion is still awaited. Terahertz spectroscopy is a powerful tool clarifying the picosecond dynamics of water molecules, and has been used for the study of hydration of various molecules in aqueous solutions. Since hydration water is usually much less mobile than that of bulk water, leading to reduced terahertz absorption, the amount of hydration water can be obtained by detecting decrease in terahertz absorption. II. RESULTS Using a high-precision terahertz time-domain spectroscopy (THz-TDS) setup [1], we have studied hydration of a protein (hen egg white lysozyme; HEWL) in aqueous solutions in the presence of various ions. In fig. 1, examples of terahertz absorption spectra observed are shown. These spectra are fea- tureless, and the magnitude of absorption coefficient depended on the species and concentration of the solutes. The absorption decrease observed in the HEWL solutions is mostly due to decrease in the amount of water in the solutions, since the proteins have terahertz absorption much less than that of water. By taking this effect into account by measuring the density of 0.2 0.4 0.6 0.8 1.0 1.2 100 150 200 250 300 Absorption Coefficient α (cm -1 ) Frequency (THz) Pure water HEWL 202 mg/mL HEWL 200 mg/mL (NH 4 ) 2 SO 4 0.25 mol/L (NH 4 ) 2 SO 4 0.25 mol/L Fig. 1. Terahertz absorption spectra of pure water, HEWL aqueous solu- tion, (NH 4 ) 2 SO 4 aqueous solution, and HEWL-(NH 4 ) 2 SO 4 mixed aqueous solution. 250 Absorption Coefficient (cm ) α −1 200 210 220 230 240 190 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Salt Concentration (mol/L) HEWL + NH 4 SCN HEWL + NH 4 H 2 PO 4 HEWL + NH 4 Cl HEWL + (NH 4 ) 2 SO 4 HEWL + NH 4 NO 3 NH 4 Cl NH 4 H 2 PO 4 NH 4 NO 3 NH 4 SCN (NH 4 ) 2 SO 4 @1 THz Fig. 2. Salt concentration dependence of absorption coefficient of salt aqueous solutions (upper half) and of HEWL(200 mg/mL)-salt mixed aqueous solutions (lower half) at 1 THz, shown with linear fitting lines. the solutions, the amount of hydration water can be estimated [2]. The salt concentration dependence of absorption coefficient at 1 THz are summarized in fig. 2. It was confirmed, by means of Fourier-transform IR and eye observation, that no denaturation, aggregation, or precipitation of HEWL took place in the observation range of concentration of each salt.