Photon Factory Activity Report 2016 #34 (2017)

BL-10C/2016G560

Effect of ultra-fine bubble water on the structure of protein and lipid membrane
1Mitsuhiro Hirai*, Satoshi Ajito, Kosuke Takahashi, Kenta Timita, and Yugo Maezawa.
Graduate School of Science and Technology, Gunma University, Maebashi, 371-8510, Japan.

1. Introduction
Ultra-fine bubble (UFB) is usually defined as small
bubbles with the diameter less than 1 µm, which isdifferent
frommicrobubbles
generallyhaving
approximately 1 ~ 100 µm in diameter. UFB is colorlessand transparent to the naked eye, and is stable for several
weeks or so in solutions. UFB enables to entrap various
kinds of gases for a long time. When oxygen is asentrapped gas, the amount of dissolved oxygen can beincreased efficiently. Unlike chemicals, it can contributeto promote the growth promote of organisms. It is said
that UFB acts on various physiological functions in living
bodies to improve the function. Even the approach to useof UFBs as DDS is now progressing.
Therefore, recently, the industrial applications of UFB
have been focused and highlighted in the various fields
such as agricultural and fishery industries, and medicaltherapy. However, the detailed mechanism underlying theperformance of UFB, especially the effect of UFB on
biological materials, is not known.
Then, we investigated the effect of UFB on protein andlipid membrane structures by using small-and-wide angle
X-ray scattering (SWAXS). The present experiment
would be the first study on the effect of UFB on
biological materials in the world as far as we know.

3. Results and Discussion
After the mixing of the protein or liposome solution
with the UFB solution at the volume ratio of 1/5, thetime-resolved SWAXS measurement was started under anappropriate time-interval. Fig. 1 shows the time evolutionof the scattering curves of LUV ([GM1] / [cholesterol] /[DMPC] = 0.1/0.1/1). The broad hump at q = ~0.1 Å-1
and the sharp peak at q = ~1.46 Å-1 reflect the lipid
bilayer structure and the alkychain packing and its
fluctuation [1, 2]. As shown clearly, we did not recognize
any evident change of the lipid bilayer structure. However,
we found significant changes of the inter-alkychain
distnace and its fluctuation. As shown in Fig. 2, both alkychain packing and chain fluctuation of lipids at gel phasein liposome tend to be suppressed in O2-UFB with in N2
bubble and pure buffer. The detailed results will be seenelsewhere.

2. Experimental
In the present study, we have investigated the effectof two different types of UFB on the proteins and theliposomes. UFB used was the ultra-high-density UFB
occluding O2 or N2 gas (10 billion particles / mL or more)offered by NANOX Co. The proteins were myoglobinfrom equine skeletal muscle, bovine serum albumin, andlysozyme from chicken egg-white, which were purchasedfrom SIGMA co. The liposome was the large uni-lamellar
vesicle (LUV) or small uni-lamellar vesicle (SUV)
composed of lipid mixture (ganglioside (GM1), cholesterol,and phospholipid (DMPC, DPPC, DOPC, POPC). where
the phospholipid concentration was fixed at 1 %w/v. The
time-resolved SWAXS measurements were carried out by
using the X-ray scattering spectrometer installed at
BL10C beam port of PF at KEK, Tsukuba, Japan. The Xray wavelengths were 1.0 Å and 1.5 Å. The sample-todetector distances were 50 cm and 300 cm. X-ray
scattering intensity was recorded by a hybrid pixel
detector, PILATUS3 2M (Dectris). The exposure timewas 60 seconds. Sample cells composed of a pair of thinquartz windows with 1 mm path length were used.

Fig. 1. Time evolution of the scattering curve of LUV([GM1]/[cholesterol]/[DMPC]=0.1/0.1/1) after dispersed
into pure water (on the left) and N2-UFB solution (on theright).

Fig. 2. Time evolution of the position (on the right) andHWHM (on the left) of the peak at ~ 1.4-1.5 Å-1 in Fig. 1.
References[1] M. Hirai et. al., J. Phys. Chem. B, 119, 3398 (2015).
[2] T. Onai & M. Hirai, J. Phys. Conference Series, 247,01208 (2010).
* mhirai@gunma-u.ac.jp