Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase

Aggregation of 3 M guanidine hydrochloride denatured creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) occurs after dilution into the refolding solution. Proline, glycerol and heparin sodium act as folding aids which can effectively inhibit aggregation of creatine kinase during refold...

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Bibliographic Details
Published in:The international journal of biochemistry & cell biology 2001-07, Vol.33 (7), p.701-709
Main Authors: Meng, Fan-Guo, Park, Yong-Doo, Zhou, Hai-Meng
Format: Article
Language:English
Subjects:
Aggregation
Anilino Naphthalenesulfonates - metabolism
Animals
Circular Dichroism
Creatine kinase
Creatine Kinase - chemistry
Creatine Kinase - metabolism
Dimerization
Folding aids
Glycerol
Glycerol - pharmacology
Heparin
Heparin - chemistry
Heparin - metabolism
Heparin - pharmacology
Muscle, Skeletal - enzymology
Proline
Proline - pharmacology
Protein Denaturation
Protein Folding
Protein Structure, Secondary
Protein Structure, Tertiary
Rabbits
Reactivation
Refolding
Spectrometry, Fluorescence
Temperature
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Summary:Aggregation of 3 M guanidine hydrochloride denatured creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) occurs after dilution into the refolding solution. Proline, glycerol and heparin sodium act as folding aids which can effectively inhibit aggregation of creatine kinase during refolding. Proline at 1 M concentration, glycerol at 10% concentration and heparin at 25 mg/ml not only completely prevented creatine kinase aggregation but also enabled the creatine kinase to return to its native state as well as to recover most of its native activity. The reactivity after the aggregation was completely blocked by the presence of each folding aid reached 65–80% of the native activity. Results of turbidity, activity, intrinsic fluorescence and 1-anilinonaphthalene-8-sulfonate binding fluorescence measurements suggested that the effect of heparin differs from that of proline and glycerol in its artificial chaperone-like behavior. Heparin may bind with creatine kinase both in the native state and during the refolding course. The results showed that this heparin–creatine kinase complex favorably restored the creatine kinase reactivity.
ISSN:1357-2725
1878-5875
DOI:10.1016/S1357-2725(01)00048-6