Human pre-alpha-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain.
Pre-alpha-inhibitor (P alpha I) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing P alpha I. Then, taking advantage of the differential affinity of the members of the inter-alpha-inhibitor family (I alpha I) for heparin-Sepharose and blue-Sepharose, we isolated P alpha I. Its specific antitryptic activity was 580 IU/g, higher than that of I alpha I: 420 IU/g. Its M(r), determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130,000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from P alpha I by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the HI and H2 heavy chains of I alpha I. In reducing conditions, it was quite similar to that of H2 (M(r) 85,000) but clearly different from that of H1 (M[r] 78,000). Thus, the so-determined apparent M(r) of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74,100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69,454) are really occupied by two N-glycans, probably of biantennary type.
Mizon
C
C
Laboratoire de Biochimie (DRED E.A. 1052), Faculté de Pharmacie, Lille, France.
Héron
A
A
Capon
C
C
Sautiere
P
P
Michalski
C
C
Sesboüé
R
R
Mizon
J
J
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015394:Molecular Structure; D011498:Protein Precursors / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D014361:Trypsin Inhibitors / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*
D011498:Protein Precursors; D014361:Trypsin Inhibitors; C061151:pre-alpha-trypsin inhibitor