実験報告書様式（一般利用課題・成果公開利用）

MLF Experimental Report

提出日 Date of Report
2011/4/13

課題番号 Project No.

装置責任者 Name of responsible person

2010B0044

Ichiro Tanaka

実験課題名 Title of experiment

装置名 Name of Instrument/（BL No.）

Neutron Crystallographic Study of Protonation states of human

iBIX/(BL03)

hemoglobin

実施日 Date of Experiment

実験責任者名 Name of principal investigator

2011/02/07-2011/02/19

Toshiyuki Chatake
所属 Affiliation
Research Reactor Institute, Kyoto University

試料、実験方法、利用の結果得られた主なデータ、考察、結論等を、記述して下さい。（適宜、図表添付のこと）
Please report your samples, experimental method and results, discussion and conclusions. Please add figures andtables for better explanation.
1. 試料 Name of sample(s) and chemical formula, or compositions including physical form.
(1) Adult human deoxyhemoglobin (DeoxyHb).
A crystal of deoxyhemoglobin (T-state) (Fig.1).*
P212121, a = 97.1 Å, b = 99.3 Å, c = 65.8 Å(2) Adult human CO-hemoglobin (COHb).
A crystal of CO-hemoglobin (R-state) (Fig.2_1).*
P41212, a = b =54.21 Å, c = 196.38 Å.

Fig.1 A Crystal ofdeoxy-hemoglobin(DeoxyHb).

*Crystals were grown in deuterated solution.

Fig.2
Crystalsofcarbomonoxy-hemoglobin (COHb).

2. 実験方法及び結果 （実験がうまくいかなかった場合、その理由を記述してください。）
Experimental method and results.

If you failed to conduct experiment as planned, please describe reasons.

Experimental and Results: Prior to collection of neutron diffraction data, preliminary neutron experiments
were carried out using crystals of DeoxyHb and COHb. A neutron time-of-flight image was taken for 10 h foreach crystal (02/07-02/08). Although the crystal of DeoxyHb was very huge; the size of the crystal was morethan 30 mm3, the maximum resolution of the diffraction image was about 3.0~3.5 Å (Fig. 3). Because the size ofthe crystal was approximately same as the beam size of iBIX (φ = 5 mm), the current power of J-PARC (220kW) is thought to be insufficient for neutron data collection of the present crystal of DeoxyHb. In order toovercome this problem, the improvement of the quality of this kind of crystal and/or higher power of J-PARC
would be necessary. In case of the crystal of COHb, a better diffraction image than the crystal of DeoxyHb wasobtained. We found diffractionts at 2.4 Å resolution on the diffraction image (Fig. 4). This crystal has a longc-axis (196.38 Å), which exceeds the nominally maximum length of a crystal lattice (135 Å) by ~60 Å,nevertheless the neighboring diffractions of the crystal of COHb seemed to be separated on the display of agraphic a workstation. We concluded that neutron diffraction images of the crystal of COHb could be processed,
and we collected the neutron diffraction data set of COHb crystal in the remaining time of this experiment.
12. 実験方法及び結果（つづき） Experimental method and results (continued)

Fig.3 A Diffraction
image of the crystal ofDeoxyHb.

Fig.4 A Diffraction
image of the crystal ofCOHb.

Fig.5 The layout of eighteen detectors in thepresent data collection.

Neutron data collection was carried out using the crystal of COHb. Fourteen wavelength-shifting fiber detectors
were used for the present data collection. The layout of the detectors is shown in Fig. 5. The power of J-PARC
was 220 kW during the data collection. The diameter of a collimator was 5 mm. The first data set was collectedwith the wavelength of 3.46 - 6.16 Å. Eighteen frames were taken with different directions of the crystal. Theexposure times were 10.2 h and 9.6 h for 1st - 3rd flames and 4th - 18th flames, respectively. In order to cover
the low-resolution area, the second data collection was carried out with the same setting except the exposure timeand wavelength. The exposure time of the second data collection and the wavelength were 1.02 h/flame and 5.86
- 8.55 Å, respectively. Assuming that the effective resolution of the present COHb crystal was 2.4 Å, it wasestimated that about 95% diffractions were covered in this experiment.
After the data collection of the crystal of COHb, the extent of methoxylation of COHb was measured, because
methoxylation of COHb should not occur during diffraction experiment. This measurement was carried out at achemistry laboratory in MLF building. The capillary was unsealed, and then crystal was solved in deuteratedbuffer. The solution was diluted and the absorption spectrum of the diluted solution was measured by anabsorption spectrometer. After the measurement, NaCN was added in the solution and the absorption spectrum
was measured again. There were very small differences between the two spectrums before and after the additionof NaCN, indicating that the methoxylation of COHb was negligibly small. Now, we are processing the neutron
diffraction frames to index, integrate and merge intensities.
Conclusion: In the present study, we succeeded in collecting the full data set of neutron diffraction of adulthuman carbomonoxy-hemoglobin. Hemoglobin has two conformations of the tetrameric structure, composed of
two alpha and two beta subunits. They are Relax and Tense states (R-state and T-state). In our previous study, wedetermined the neutron structure of the T-state adult human deoxyhemoglobin. On the other hand, COHb takes
R-state. It is expected that the comparison between protonation behaviors of the two states provides the insight ofthe mechanism of oxygen-transportation by hemoglobin. In addition, the present study shows the capacity ofiBIX, which supports a longer unit cell than the estimate. The nominally maximum length is 135 Å for iBIX,
meanwhile the length of the c-axis of the crystal of COHb was 196.4 Å. Neighboring diffractions around 2.4 Å
resolution can be separated in this crystal, indicating that iBIX can support a long crystal lattice (~200 Å) if theeffective resolution is medium.
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