Jia Jia, Wei Chen, Huimin Ma, Ke Wang, Chuan Zhao
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
Molecular bioSystems 2010 OctRhodamine B piperazinoacetohydrazine (RBPH) is used as a bifunctional probe for the N-terminal specific modification of a thermophilic enzyme (T. lanuginosus xylanase), and the modification effect on the thermostability of the enzyme is investigated. The probe RBPH, bearing a spectroscopic unit of rhodamine B, a carbonyl-specific labeling unit of hydrazine and a linker of piperazine, not only has a stable always-on spectroscopic response, but also exists in a cationic form. These properties enable RBPH to serve as a bifunctional probe (simultaneous introduction of stable spectroscopic signal and positive charge) for the protein modification, and such an application has been successfully demonstrated on the N-terminal labeling of T. lanuginosus xylanase. A temperature-dependent inactivation study shows that the modification of T. lanuginosus xylanase by RBPH hardly changes its thermostability, in other words, a small change in electric charge of the N-terminal region caused by introducing one positive charge is not enough to alter the thermostability of the enzyme. This reveals a conservative property of the N-terminal domain for electric charge change, and such a property may result from the fact that the N-terminal domain of the enzyme already has 4 charged residues, which can produce strong electrostatic interactions, thereby making the domain quite stable.
Jia Jia, Wei Chen, Huimin Ma, Ke Wang, Chuan Zhao. Use of a rhodamine-based bifunctional probe in N-terminal specific labeling of Thermomyces lanuginosus xylanase. Molecular bioSystems. 2010 Oct;6(10):1829-33
PMID: 20607149
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