NDLI: Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System

Content Provider	Springer Nature Link
Author	Fan, Yong Qiang Liu, Hong Jian Li, Chang Luan, Yu Shi Yang, Jun Mo Wang, Yu Long
Copyright Year	2012
Abstract	In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k $_{2}$ value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8 × 10$^{−5}$ s$^{−1}$. The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.
Starting Page	268
Ending Page	280
Page Count	13
File Format	PDF
ISSN	02732289
Journal	Applied Biochemistry and Biotechnology
Volume Number	169
Issue Number	1
e-ISSN	15590291
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-11-22
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Human brain-type creatine kinase Macromolecular crowding Guanidine Hydrochloride denature Inactivation Kinetics Biotechnology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Environmental Engineering Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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