title: Analysis of trypsin inhibition activity in human plasma proteins after separation by non-denaturing two-dimensional electrophoresis.
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.
  
  
  CONCLUSIONS
  This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the trypsin inhibition activities of proteins.
authors.affiliation: 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.
authors.forename: Youji
authors.initials: Y
authors.lastname: Shimazaki
authors.forename: Madoka
authors.initials: M
authors.lastname: Michhiro
mesh_terms: D001391:Azo Compounds; D002364:Caseins / Q000737:chemistry; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional; D006242:Haptoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008567:Membranes, Artificial; D010449:Peptide Mapping; D011145:Polyvinyls; D012709:Serum Albumin / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000302:isolation & purification; D005465:Fluorocarbon Polymers
chemical_list: D001391:Azo Compounds; D002364:Caseins; D006242:Haptoglobins; D008567:Membranes, Artificial; D011145:Polyvinyls; D012709:Serum Albumin; D014168:Transferrin; D014357:Trypsin; D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins; C026018:azocasein; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C032756:ponceau S
keywords: 2-Amino-2-hydroxymethyl-1,3-propanediol; 2-DE; 3-D map; ACTH; Adrenocorticotropic hormone; CBB; Coomassie Brilliant Blue; Haptoglobin; IEF; Isoelectric focusing; MALDI-TOF MS; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Non-denaturing; N′-tetramethylenediamine; PVDF; Polyvinylidene fluoride; TEMED,N,N,N′; Tris; Trypsin inhibition activity; Two-dimensional electrophoresis; α(2)-Macroglobulin