Microwave dielectric study on bound water of globule proteins in aqueous solution

Abstract

SYNOPSISA dielectric relaxation peak due to bound water of globule proteins in aqueous solution was observed at first by the use of a time domain reflectometry. This peak locates around 100 MHz as well as that of the aqueous DNA solution and the moist collagen, and has a relaxation strength in proportion to surface of the globule protein except for trypsin and pepsin of hydrolase. It is suggested that this peak is caused by orientation of bound water molecules on the protein surface. The number of bound water molecules estimated is in good agreement with that obtained by other method such as x-ray analysis. The solution exhibits another peak below 100 MHz, which is caused by the rotation of globule protein supplemented by migration of the counterion. Its relaxation time is completely proportional to the molecular weight of the protein. I NTRO DUCT10 NGlobule protein is one of the most important materials in biology. It has a lot of biological functions in living materials. For example, the enzyme works as a catalyst of hydrolysis, oxidation and reduction. In the living materials the protein molecules are surrounded by water in most cases. Therefore it is of particular interest to investigate the water structure around the protein.High resolution x-ray and neutron diffraction measurements in the crystal phase have made clear that there are peculiar water molecules correlating strongly with the globule protein.' It is suggested that the water molecules attach to the oxygen, nitrogen, and polar groups on the globule protein surface through hydrogen bonding. This bound water is thought to give an important influence on the functions of protein. In the case of trypsin of hydrolase, such water molecules construct a network with polar groups, which maintains the shape of active site, and stabilizes the protein structure. Existence of this water is also ascertained by a computer simulation employing molecular dynamic^.^,^ On the other hand, the sorption measurement of water vapor indicated that there are often a number of sorption points on the globule protein that is estimated, from a relationship between the vapor pressure and the amount of ~o r p t i o n .~The numbers of sorption point of albumin, cytochrome C and lysozyme obtained are 241, 56, and 60, respectively, which are roughly equal to those of polar residues per protein molecule, which are 375,65, and 80, re~pectively.~ It has been thus suggested that the sorption water is bound to the polar group^.^ However, most of the functions of globule protein does not appear in the crystal phases, where the protein structure is stabilized by the intermolecular interaction and the large structural fluctuation is restricted. Then water in the crystal phase seems to be different from that in vivo. Therefore it is again interesting to know the structure of bound water in aqueous solution.Recently dielectric measurement by using a time domain reflectometry (TDR) method have been performed on the DNA aqueous solution5 and the moist collagen.6 Two relaxation peaks were observ...

Protein Hydration Dynamics in Aqueous Solution: A Comparison of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin by Oxygen-17 Spin Relaxation Dispersion

“…For myoglobin it was concluded that half of the (several hundred) surface water molecules have t S /t bulk 1 5 (in agreement with our results), while the other half have t S /t bulk 1 1200 (Pethig, 1992). More recently, dielectric relaxation data from ten globular proteins were interpreted in terms of 50 to 250 surface water molecules per protein with an average t S /t bulk value in the range 160 to 325 (Miura et al, 1994).…”

Protein hydration dynamics in solution: a critical survey

“…To be observable against the background of bulk water (some 10 4 bulk water molecules at a protein concentration of 5 mM), the ␦ dispersion must then be attributed to a large number of hydration water molecules (in the order of 10 2 ). Many DRS studies have thus concluded that a sizeable fraction (typically, about one-half) of the water molecules in the hydration layer are strongly rotationally retarded, with ͗ hyd ͘/ bulk in the range of 10 2 -10 3 (Dachwitz et al 1989;Pethig 1992Pethig , 1995Miura et al 1994;Wei et al 1994). This conclusion is grossly inconsistent with the MRD results (see § 5a), yielding ͗ hyd ͘/ bulk Ϸ 2 for the vast majority of water molecules in the hydration layer.…”

The Formation of Protein Structure

“…In proteins of thi type, all domains are about the same size Direct confirmation of the existence wring modes using spectroscopy would b e an important test of the model. Microwa\ spectra reveal features in the correct fre quency range [12] -features that have been linked to the presence of water bound to the protein molecule, for when proteins are dehydrated the microwave absorption disappears. It is necessary to consider two alternative possibilities: a) that the disap pearance of the microwave absorption is directly related to the lack of bound water molecules; b) that the disappearance is du e to a change in the nature of the topologica l constraint of the protein molecule, i.e., the dehydrated protein is not topologically constrained at the wring mode frequency peptides.…”