| Project/Area Number |
16590033
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Josai University |
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Principal Investigator |
SHIRAHATA Akira Josai University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50150107)
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| Co-Investigator(Kenkyū-buntansha) |
SUGITA Yoshiaki Josai University, Faculty of Pharmaceutical Sciences, Lecturer, 薬学部, 講師 (20255029)
IKEGUCHI Yoshihiko Josai University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (00364711)
TAKAO Koichi Josai University, Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (70337484)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Conformational change / Fluorescent labeling agent for sulfhydryl groups / C-terminal labeling / MALDI-TOFHS / PSD / Polyamine / グアニジン / 蛍光検出HPLC / アミノ酸配列分析 |
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| Research Abstract |
In this study, firstly we developed the detection for conformational change of proteins on their binding small molecules by using a model protein. Secondly, the evaluation of the fluorescent intensity change of synthesized labeling agents for sulfhydryl group enabled the analysis for conformational change of proteins. The method also made it possible to analyze amino acid sequences in peptides labeled with the fluorescent labeling agent for sulfhydryl group by MALDI-PSD TOF MS, following fractionation with high performance liquid chromatography (HPLC) for the fluorescence-labeled proteins digested by protease.
Two similar molecular fluorescent agents for sulfhydryl group were synthesized, and the combination of the two agent and MALDI-TOF MS enabled the evaluation for conformational changes of proteins, by comparison of relative signal intensity ratio of between the peptides digested following label of the two agents respectively.
On the other hand, the novel labeling method for C-terminal labeling in the peptides during cleavage of proteins was developed. This method is found to be applicable for labeling by various alkylamines with some charges, and for structural analysis, and also for comparison determination with MALDI-TOF MS.
Furthermore, the effect of polyamines on the digestion of proteins by serine proteases was examined. Hemoglobin was used as a model protein and was digested with trypsin in the presence of polyamine. The product peptides were separated, collected by HPLC, and analyzed by MALDI-TOF MS using post-source decay. The results showed that some peptides were indeed modified with polyamine at the C-terminus.
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