Naslov
Preparation of highly purified Inter-alpha inhibitor proteins from human plasma and their analysis by use of SELDI-TOF technology.

Autori
M.K. Brown, Y-P. Lim, M. Ručević, M. Kim, D.C. Hixson, D. Josić.

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
COBRE CCRD Research Symphosium

Mjesto i datum
Providence (RI), Sjedinjene Američke Države, 24.06.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
Inter-alpha inhibitor proteins; chromatographic methods; SSELDI-TOF technique; SDS-PAGE

Sažetak
Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors (serpins) found in relatively high concentrations in human plasma. The major forms found in human plasma are inter-alpha inhibitor (IaI), which consists of two heavy chains (H1 & H1) and a single light chain (bikunin), and pre alpha inhibitor (PaI), which consists of one heavy (H3) and one light chain. Recently, the involvement of IaIp in sepsis has become an area of intensive investigation. We found markedly decreased plasma levels of IaIp in septic patients compared to healthy controls. Furthermore, the concomitant increase of IaIp-related fragments in uine suggest that these proteins are “ consumed” and rapidly cleared from the systemic circulation during sepsis. This offers a therapeutic rationale for replacement of IaIp during sepsis. Using a combination of several chromatographic methods, both members of IaIp family (IaIp and PaI) were isolated from human plasma in highly purity and biologically active forms. The highly purified IaIp preparation was effective in reducing mortality in the experimental animal model. For characterization of the isolated proteins and identification of the main contaminant during purification we used SELDI-TOF technique. After separation by SDS-PAGE, polypeptide bands with apparent molecular weights of 80 kDa, 125 kDa, and 25 kDa were cut out from the gel, reduced, akylated, and digested by trypsin. After extraction, the peptides were spotted to a hydrophobic and SEDN-ID chip and analyzed by mass spectrometry. To our surprise, the 80 kDa band was identified as heavy chain H3 precursor. Another plasma protein, clotting factor X, which was identified in this band by Western blot and measurement of clotting activity seems to be only a minor component in the fraction. In the 125 kDa band containing PaI not only heavy chain H3, but also heavy chains H1, H2 and recently described heavy chain H4 together with bikunin were detected. The 250 kDa band contains only two heavy chains H1 and H2 together with the light chain, bikunin. Heavy chains H3 and H4 were not detected in this band. Current efforts are focused on further biochemical and clinical investigation of these preparation as an effective strategy for reducing the severe complications of sepsis in humans.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biotehnologija

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