Jun 9, 1995

Thermal stability and domain-domain interactions in natural and recombinant protein C

The Journal of Biological Chemistry
L V MedvedK C Ingham

Abstract

Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins be...Continue Reading

Mentioned in this Paper

Epidermal Growth Factor
Calcium
Protein C, human
Proteins, Recombinant DNA
Osteocalcin
Fluorescence
Protein C Measurement
Protein C Antigen Measurement
Serine Endopeptidases
PROC gene

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