Formation of intraprotamine disulfides in vitro.
When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solubilization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein's cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the amino- and carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro.
Balhorn
R
R
Biomedical Division, Lawrence Livermore National Laboratory, Livermore, California 94550.
Corzett
M
M
Mazrimas
J A
JA
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004589:Electrophoresis, Disc; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D011479:Protamines / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012661:Semen / Q000737:chemistry; D013552:Swine
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D011479:Protamines; D008623:Mercaptoethanol; D004247:DNA; D004229:Dithiothreitol