Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

摘要: We characterized the biochemical functions of small nonessential (C101–C104) and large essential (C173–C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] [brDNase(C173A) brDNase(C209A)], respectively. also effects an additional third disulfide [brDNase(F192C/A217C)]. Without Ca2+ protection, bpDNase brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca2+, all forms DNase, except for brDNase(C101A), protected against trypsin. All after being dissolved 6 M guanidine-HCl, fully reactivated diluting into a Ca2+-containing buffer. However, when diluted Ca2+-free buffer, inactive, but 60% activity was restored with brDNase(F192C/A217C). When heated, at transition temperature 65°C, 60°C, 73°C, indicating that disulfide, albeit not activity, is important structural integrity, introduction can further stabilize enzyme. pellets brDNase(C173A) brDNase(C209A) inclusion bodies guanidine-HCl then 10%–18% restored, suggesting “essential” absolutely crucial enzymatic catalysis. Owing to structure-based sequence alignment revealing homology between “nonessential” active-site motif thioredoxin, we measured 39% thioredoxin-like based on rate insulin precipitation (ΔA650nm/min). Thus, only play role stabilizing protein molecule may engage biological such as disulfide/dithiol exchange reaction.