title: Measuring the bioactivity and molecular conformation of typically globular proteins with phenothiazine-derived methylene blue in solid and in solution: A comparative study using photochemistry and computational chemistry.
abstract: Methylene blue is a phenothiazine agent, that possesses a diversity of biomedical and biological therapeutic purpose, and it has also become the lead compound for the exploitation of other pharmaceuticals such as chlorpromazine and the tricyclic antidepressants. However, the U.S. Food and Drug Administration has acquired cases of detrimental effects of methylene blue toxicities such as hemolytic anemia, methemoglobinemia and phototoxicity. In this work, the molecular recognition of methylene blue by two globular proteins, hemoglobin and lysozyme was characterized by employing fluorescence, circular dichroism (CD) along with molecular modeling at the molecular scale. The recognition of methylene blue with proteins appears fluorescence quenching via static type, this phenomenon does cohere with time-resolved fluorescence lifetime decay that nonfluorescent protein-drug conjugate formation has a strength of 10(4)M(-1), and the primary noncovalent bonds, that is hydrogen bonds, π-conjugated effects and hydrophobic interactions were operated and remained adduct stable. Meantime, the results of far-UV CD and synchronous fluorescence suggest that the α-helix of hemoglobin/lysozyme decreases from 78.2%/34.7% (free) to 58.7%/23.8% (complex), this elucidation agrees well with the elaborate description of three-dimensional fluorescence showing the polypeptide chain of proteins partially destabilized upon conjugation with methylene blue. Furthermore, both extrinsic fluorescent indicator and molecular modeling clearly exhibit methylene blue is situated within the cavity constituted by α1, β2 and α2 subunits of hemoglobin, while it was located at the deep fissure on the lysozyme surface and Trp-62 and Trp-63 residues are nearby. With the aid of computational analyses and combining the wet experiments, it can evidently be found that the recognition ability of proteins for methylene blue is patterned upon the following sequence: lysozyme<hemoglobin<albumin. Basically, the distinction originates from different spatial structures of proteins and noncovalent interactions between proteins and methylene blue. In addition, biological relevance of the biorecognition of methylene blue with proteins was briefly discussed. We hope that this study could provide further standpoint so that one explore the biological activity of methylene blue and also phenothiazines.
authors.affiliation: College of Agriculture and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China; Department of Chemistry, China Agricultural University, Beijing 100193, China; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Ding
authors.affiliation: State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Research Institute of Chemical Industry Co. Ltd., Shenyang 110021, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Xie
authors.affiliation: College of Agriculture and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China; College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China. Electronic address: weipeng@cau.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Peng
authors.affiliation: Center for Food Quality Supervision & Testing, Ministry of Agriculture, College of Food Science & Engineering, Northwest A&F University, Yangling 712100, China.
authors.forename: Yu-Kui
authors.initials: YK
authors.lastname: Peng
mesh_terms: D002942:Circular Dichroism; D006801:Humans; D008751:Methylene Blue / Q000737:chemistry*; D010640:Phenothiazines / Q000737:chemistry*; D010777:Photochemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions
chemical_list: D010640:Phenothiazines; D011506:Proteins; D012996:Solutions; C031637:phenothiazine; D008751:Methylene Blue
keywords: Globular proteins; Methylene blue; Molecular modeling; Phenothiazine; Photochemistry