Abstract
Human low-molecular-weight kininogen (LMWK) was purified to apparent physical and functional homogeneity by a six-step procedure consisting of ion-exchange chromatography, reverse ammonium sulfate gradient solubilization, hydrophobic chromatography on phenyl-Sepharose, gel filtration, and removal of contaminating proteins by their affinity for Affi-Gel blue and zinc. The recovery averaged 15.6% ( n = 4). Purified LMWK presented as a single stained band on alkaline polyacrylamide gel electrophoresis which corresponded to the region of function in eluates from a duplicate gel. The apparent homogeneity was also observed in sodium dodecyl sulfate (SDS)-gel electrophoresis, where the protein presented as a single band of M r = 65,000 without reduction and 68,000 with reduction. A mole of substrate released 0.8 mol of kinin in 5 min when cleaved by human urinary kallikrein (HUK), and 0.9 mol after 30 min. Cleavage of the single-chain LMWK released kinin from within a disulfide loop as indicated by the SDS-gel electrophoresis of reduced and unreduced kinin-free LMWK. The heavy chain exhibited an M r = 62,000, which is similar to the M r of the amino-terminal chain of human HMWK and is consistent with their antigenic relatedness. In contrast to the M r = 64,000 procoagulant chain of human HMWK, the small (<10,000) carboxy-terminal chain of LMWK has no procoagulant activity and may serve only to protect the kinin moiety in the intact substrate.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.