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Protein Science (2004), 13:875-883. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

Wei-Jung Chen, I-Shuan Lee, Ching-Ying Chen and Ta-Hsiu Liao

Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan

(RECEIVED September 15, 2003; FINAL REVISION December 29, 2003; ACCEPTED January 7, 2004)



Abstract

We characterized the biochemical functions of the small nonessential (C101–C104) and the large essential (C173–C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca2+, all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca2+-containing buffer. However, when diluted into a Ca2+-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65°C, brDNase(C101A) at 60°C, and brDNase(F192C/A217C) at 73°C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca2+-containing buffer, 10%–18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation ({Delta}A650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.

Keywords: deoxyribonuclease; cysteine; disulfide; site-directed mutagenesis; thioredoxin; thermal stability; protein refolding

Abbreviations: bp, bovine pancreatic • br, bovine recombinant • brDNase (F192C/A217C), the brDNase double mutant with changes of Phe192 to Cys192 and Ala217 to Cys217 • brDNase(C101A), the brDNase mutant with change of Cys101 to Ala101 • brDNase(C173A), the brDNase mutant with change of Cys173 to Ala173 • brDNase(C209A), the brDNase mutant with change of Cys209 to Ala209 • SDS, sodium dodecyl sulfate • PAGE, polyacrylamide gel electrophoresis • cm-Cys, S-carboxymethyl Cys

Reprint requests to: Ta-Hsiu Liao, Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei 10018, Taiwan; e-mail: thliao{at}ccms.ntu.edu.tw; fax: 886-2-2394-6747.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03438204.


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