Analysis of RNase A refolding intermediates by electrospray/mass spectrometry.
Electrospray/mass spectrometry (ES/MS) was extensively used to obtain information on disulphide-containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular S-S are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides. The results indicate that ES/MS is a suitable technique to detect and characterize refolding intermediates.
Torella
C
C
Servizio Spettrometria di Massa, Naples, Italy.
Ruoppolo
M
M
Marino
G
G
Pucci
P
P
D000818:Animals; D002417:Cattle; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D013058:Mass Spectrometry; D010179:Pancreas / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013997:Time Factors; D014508:Urea
D006146:Guanidines; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D014508:Urea; D012259:Ribonuclease, Pancreatic; D019791:Guanidine