Elsevier

Analytical Biochemistry

Volume 134, Issue 2, 15 October 1983, Pages 336-346
Analytical Biochemistry

Purification of single-chain human low-molecular-weight kininogen and demonstration of its cleavage by human urinary kallikrein

https://doi.org/10.1016/0003-2697(83)90307-XGet rights and content

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 Mr = 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 Mr = 62,000, which is similar to the Mr of the amino-terminal chain of human HMWK and is consistent with their antigenic relatedness. In contrast to the Mr = 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.

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    Supported by Grants HL-22939, AI-07722, AI-10356, AM-05577, and RR-05669 from the National Institutes of Health.

    2

    Postdoctoral fellow of the Max Kade Foundation, Inc. Present address: Institute of Medical Physiology, Medical Faculty of the University of Vienna, Vienna A-1090, Austria.

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