Use of a rhodamine-based bifunctional probe in N-terminal specific labeling of Thermomyces lanuginosus xylanase.
Rhodamine 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
J
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
Chen
Wei
W
Ma
Huimin
H
Wang
Ke
K
Zhao
Chuan
C
D001203:Ascomycota / Q000201:enzymology*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D015335:Molecular Probes; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D015335:Molecular Probes; D012235:Rhodamines; D043364:Endo-1,4-beta Xylanases