Youji Shimazaki1, Madoka Michhiro. 1. Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan. Electronic address: shimazaki.yoji.my@ehime-u.ac.jp.
Abstract
BACKGROUND: The functions of proteins can be retained following separation by non-denaturing two-dimensional electrophoresis (2-DE). The trypsin inhibition activities can then be examined following the separation and immobilization of the proteins under non-denaturing conditions. METHODS: Human plasma proteins were separated using 2-DE and transferred onto a polyvinylidene difluoride membrane and stained using Ponceau S. The trypsin inhibition activity of the membrane-bound proteins was qualitatively examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The activities were also quantitatively examined by analyzing the release of the azo-chromophore when azocasein was the substrate. RESULTS: Trypsin activity was inhibited by the haptoglobin and α2-macroglobulin spots located on the membrane, whereas the protease activity was retained for the spots containing albumin and transferrin. The inhibition activities of the α2-macroglobulin and haptoglobin spots were 4.81- and 4.83-fold higher, respectively, when compared with the inhibition activity of the albumin spot. An axis of the relative activities of trypsin inhibition was added to the 2-DE pattern of human plasma proteins to construct a non-denaturing 3-D map of human plasma proteins. CONCLUSION: This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the trypsin inhibition activities of proteins.
BACKGROUND: The functions of proteins can be retained following separation by non-denaturing two-dimensional electrophoresis (2-DE). The trypsin inhibition activities can then be examined following the separation and immobilization of the proteins under non-denaturing conditions. METHODS:Human plasma proteins were separated using 2-DE and transferred onto a polyvinylidene difluoride membrane and stained using Ponceau S. The trypsin inhibition activity of the membrane-bound proteins was qualitatively examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The activities were also quantitatively examined by analyzing the release of the azo-chromophore when azocasein was the substrate. RESULTS: Trypsin activity was inhibited by the haptoglobin and α2-macroglobulin spots located on the membrane, whereas the protease activity was retained for the spots containing albumin and transferrin. The inhibition activities of the α2-macroglobulin and haptoglobin spots were 4.81- and 4.83-fold higher, respectively, when compared with the inhibition activity of the albumin spot. An axis of the relative activities of trypsin inhibition was added to the 2-DE pattern of human plasma proteins to construct a non-denaturing 3-D map of human plasma proteins. CONCLUSION: This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the trypsin inhibition activities of proteins.