High-permeability alternatives to current dialyzers performing both high-flux hemodialysis and postdilution online hemodiafiltration.

Most high-flux dialyzers can be used in both hemodialysis (HD) and online hemodiafiltration (OL-HDF). However, some of these dialyzers have higher permeability and should not be prescribed for OL-HDF to avoid high albumin losses. The aim of this study was to compare the safety and efficacy of a currently used dialyzer in HD and OL-HDF with those of several other high permeability dialyzers which should only be used in HD. A prospective, single-center study was carried out in 21 patients. Each patient underwent 5 dialysis sessions with routine dialysis parameters: 2 sessions with Helixone (HD and postdilution OL-HDF) and 1 session each with steam sterilized polyphenylene, polymethylmethacrylate (PMMA), and medium cut-off (MCO) dialyzers in HD treatment. The removal ratios (RR) of urea, creatinine, ß2 -microglobulin, myoglobin, prolactin, α1 -microglobulin, α1 -acid glycoprotein, and albumin were compared intraindividually. A proportional part of the dialysate was collected to quantify the loss of various solutes, including albumin. Urea and creatinine RRs with the Helixone-HDF and MCO dialyzers were higher than with the other 3 dialyzers in HD. The ß2 -microglobulin, myoglobin and prolactin RRs with Helixone-HDF treatment were significantly higher than those obtained with all 4 dialyzers in HD treatment. The ß2 -microglobulin value obtained with the MCO dialyzer was also higher than that obtained with the other 3 dialyzers in HD treatment. The myoglobin RR with MCO was higher than those obtained with Helixone and PMMA in HD treatment. The prolactin RR with Helixone-HD was significantly lower than those obtained in the other 4 study sessions. The α1 -microglobulin and α1 - acid glycoprotein RRs with Helixone-HDF were significantly higher than those obtained with Helixone and PMMA in HD treatment. The albumin loss varied from 0.54 g with Helixone-HD to 3.3 g with polyphenylene. The global removal score values ((UreaRR + ß2 -microglobulinRR + myoglobinRR + prolactinRR + α1 -microglobulinRR + α1 -acid glycoproteinRR - albuminRR )/6) were 43.7% with Helixone-HD, 47.7% with PMMA, 54% with polyphenylene, 54.8% with MCO and 59.6% with Helixone-HDF, with significant differences. In conclusion, this study confirms the superiority of OL-HDF over HD with the high-flux dialyzers that allow both treatments. Although new dialyzers with high permeability can only be used in HD, they are in an intermediate position and some are very close to OL-HDF.

Medium Cut-Off Dialyzer versus Eight Hemodiafiltration Dialyzers: Comparison Using a Global Removal Score.

BACKGROUND: A novel class of membranes, medium cut-off (MCO) membranes, has recently been designed to achieve interesting removal capacities for middle and large middle molecules in hemodialysis (HD) treatments. The few studies published to date have reported contradictory results regarding middle-sized molecules when comparing MCO dialyzers versus dialyzers used in online hemodiafiltration (OL-HDF). METHODS: A prospective, single-center study was carried out in 22 patients. Each patient underwent 9 dialysis sessions with routine dialysis parameters, one with an MCO dialyzer in HD and the other 8 with different dialyzers in OL-HDF. The removal ratio (RR) of urea, creatinine, ß2-microglobulin, myoglobin, prolactin, α1-microglobulin, α1-acid glycoprotein, and albumin was intraindividually compared. Albumin loss in dialysate was measured. We propose a global removal score ([ureaRR + ß2-microglobulinRR + myoglobinRR + prolactinRR + α1-microglobulinRR + α1-acid glycoproteinRR]/6 - albuminRR) as a new tool for measuring dialyzer effectiveness. RESULTS: No significant differences in the RRs of small and middle molecular range molecules were observed between the MCO vs. OL-HDF dialyzers (range 60-80%). Lower RRs were found for α1-microglobulin and α1-acid glycoprotein without significant differences. The albumin RR was < 11% and dialysate albumin loss was < 3.5 g in all situations without significant differences. The global removal score was 54.9 ± 4.8% with the MCO dialyzer without significant differences. CONCLUSIONS: Removal of a wide range of molecular weights, calculated with the proposed global removal score, was almost equal with the MCO dialyzer in HD treatment compared with 8 high-flux dialyzers in high-volume OL-HDF without relevant changes in albumin loss. The global removal score could be a new tool to evaluate the effectiveness of dialyzers and/or different treatment modalities.

Purification and determination of C-reactive protein and inter-α-trypsin inhibitor heavy chain 4 in dogs after major surgery through generation of specific antibodies.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) and C-reactive protein (CRP) have been isolated from acute phase dog sera by affinity chromatography with insolubilized polyclonal antibodies anti pig Major Acute phase Protein (Pig-MAP) and with p-Aminophenyl Phosphoryl Choline, respectively. Isolated proteins were used to prepare specific polyclonal rabbit antisera that have allowed quantifying their concentration in serum samples by single radial immunodifussion. Both proteins were quantified in sera from female dogs that had undergone ovariohysterectomy (OVH, n=9) or mastectomy (n=10). The observed increases in CRP concentrations showed that surgical traumas induced an acute phase response of a great magnitude in the dogs. In both surgeries a four-fold increase of ITIH4 concentrations was detected. It can be concluded that ITIH4 is a new positive acute phase protein in dogs, as reported in other species.

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations.

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor.

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex.

The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAß3Galß3Galß4Xylß-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcß4GlcAß3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.

Sulfation of the bikunin chondroitin sulfate chain determines heavy chain·hyaluronan complex formation.

Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 M NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1-1.0 M NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5-0.8 M NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1-0.5 M NaCl) contained unsulfated and 4-sulfated CS disaccharides. IαI in the 0.5-0.8 M NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1-0.5 M NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.

Evaluation of extraction solutions for biochemical analyses of the proteins in rice grains.

The influence of different extraction solutions on the proteins extracted from rice grains was investigated. The largest amounts of salt-soluble proteins were extracted with solutions supplemented with Tris-HCl at pH 8.0. Rice allergens were analyzed by multiplex immunodetection. Except for α-globulin extracted with the solutions at pH 8.0, which showed a low-molecular-weight band besides the main band, no significant solution-dependent difference among the allergens was found. Total proteins were extracted with four kinds of solution. The extraction of the basic subunit of glutelin was found to be SDS-dependent, and more protein was obtained with extraction solutions supplemented with SDS. The contents of α-globulin and α-amylase/trypsin inhibitors were higher in the extracts without SDS than with SDS. We conclude from the present data that, in order to obtain comparable data from rice grain salt-soluble and total protein analyses, differences in the protein extraction efficiency of solutions used should be taken into consideration.

Cloning, purification, crystallization and preliminary X-ray studies of human α1-microglobulin.

α(1)-Microglobulin (α(1)m) is one of the phylogenetically most widespread lipocalins and is distributed in various organs and tissues, including liver, heart, eye, kidney, brain, lung, pancreas and skeletal muscle. α(1)m has been found to exert multifarious functions, including interacting with IgA, albumin and prothrombin, binding strongly to haem and exhibiting reductase activity. Nevertheless, little structural information is available regarding these functions of α(1)m. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography was used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of human α(1)m are reported. The crystal belonged to space group P4(3), with unit-cell parameters a = b = 36.45, c = 112.68 Å, and diffracted to a resolution of 2.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 1.63 Å(3) Da(-1).

Growth factor- and adhesion protein-like components of fetal calf serum can significantly enhance the intracellular delivery of Peptide nucleic acids.

Evidence is presented that components of fetal calf serum (FCS) can significantly enhance the splicing correction activity of peptide nucleic acids (PNA) in HeLa pLuc 705 cells. The effect proved more pronounced for PNAs bearing fluorescence tags and relies on the ability of specific components of FCS to mediate a mainly nonendocytotic intracellular delivery of PNA. Attempts to isolate and characterize the active serum components using PNA-loaded beads and nano-LC-ESI mass spectrometry revealed the growth-factor related inter-alpha-trypsin inhibitor and the adhesion protein fibronectin to be substantially responsible for the delivery activity of FCS.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry.

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats. This novel alpha2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha2u. The present investigation confirms the presence of alpha2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.

Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-alpha-inhibitor (HC*HA) purified from extracts of human amniotic membrane.

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of approximately 3000 kDa) was predominantly extracted in isotonic Extract A (70.1 +/- 6.0%) and PBS (37.7 +/- 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-alpha-inhibitor (IalphaI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-alpha stimulated gene-6 protein (TSG-6). This HC.HA complex (nHC*HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC*HA) by mixing HMW HA, serum IalphaI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-beta1 promoter activation in corneal fibroblasts and induced mac ro phage apoptosis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC*HA or rcHC*HA. These data collectively suggest that the HC*HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.

Expression and purification of active recombinant human bikunin in Pichia pastoris.

Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification procedure. The cDNA encoding human bikunin was cloned by PCR and inserted into the expression vector pPICZalphaC. After expressed in shake flask, rh-bikunin was produced in an 80-L fermenter and purified by cation exchange chromatography and reverse phase chromatography. The rh-bikunin was active by trypsin inhibition test. The final expression levels were 55 mg/L and we got totally 1.44 g (5600 inhibitor units/mg) of purified rh-bikunin (purity is 95%) from 40 L of fermentation broth. The rh-bikunin consists of two forms with molecular masses of 24 and 21 kDa, respectively. Both forms were immunoreactive by Western blotting and N-terminals were correctly processed by amino-terminal sequencing. This study provided a new method for expression and purification of active rh-bikunin.

Analysis of immune complexes by capillary electrophoresis.

A simple method for immune complexes (IC) analysis by CE is described. This method combines the ease of precipitation of the IC by polyethylene glycol with the separation power of CE. The advantage of this method is a better quantitation of the IC, since it corrects and eliminates the interferences from other serum proteins. It also reveals the composition (monoclonality) of the precipitate. Three types of IC have been detected in this method: monoclonal, polyclonal and mixed (mono-polyclonal) IC. Furthermore, the method is rapid and simple.

The lipocalin alpha1-microglobulin has radical scavenging activity.

The lipocalin alpha(1)-microglobulin (alpha(1)m) is a 26-kDa glycoprotein present in plasma and in interstitial fluids of all tissues. The protein was recently shown to have reductase properties, reducing heme-proteins and other substrates, and was also reported to be involved in binding and scavenging of heme and tryptophan metabolites. To investigate its possible role as a reductant of organic radicals, we have studied the interaction of alpha(1)m with the synthetic radical, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS radical). The lipocalin readily reacted with the ABTS radical forming reduced ABTS. The apparent rate constant for this reaction was 6.3 +/- 2.5 x 10(3) M(-1) s(-1). A second reaction product with an intense purple color and an absorbance maximum at 550 nm was formed at a similar rate. This was shown by liquid chromatography/mass spectrometry to be derived from covalent attachment of a portion of ABTS radical to tyrosine residues on alpha(1)m. The relative yields of reduced ABTS and the purple ABTS derivative bound to alpha(1)m were approximately 2:1. Both reactions were dependent on the thiolate group of the cysteine residue in position 34 of the alpha(1)m polypeptide. Our results indicate that alpha(1)m is involved in a sequential reduction of ABTS radicals followed by trapping of these radicals by covalent attachment. In combination with the reported physiological properties of the protein, our results suggest that alpha(1)m may be a radical reductant and scavenger in vivo.

[Luminescent analysis of the structure of human alpha-1-microglobulin].

The spectra of absorption, fluorescence, and excitation of fluorescence of preparations of alpha-1-microglobulin isolated from human urea by two methods, gel chromatography and immunoaffinity chromatography with additional purification by activated charcoal, have been investigated in ultraviolet and visible regions. A possible nature of low-molecular compounds coloring alpha-1-microglobulin yellow-brown and their role in stabilizing the structure of protein globule are discussed. The action of urea (1.0-10 M) and guanidine hydrochloride (0.25-6 M) on the conformational state and the fast (nanosecond) internal dynamics of alpha-1-microglobulin has been investigated by the method of tryptophan fluorescence. It has been shown that the unfolding of alpha-1-microglobulin under the action of these denaturants is associated with a significant increase in the nanosecond internal dynamics of protein. The ability of alpha-1-microglobulin to restore the initial conformation characteristic for the native protein and the internal dynamics after the unfolding of the globule by 10 M urea and 6 M guanidine hydrochloride has been ascertained. It has been found that alpha-1-microglobulin isolated by the method of gel chromatography can exist in solution of 4-6 M urea in a thermodynamically stabile partialy folded state.

SAXS studies of human protein HC (alpha1-microglobulin).

Protein HC is a low molecular weight heterogeneous glycoprotein widely distributed in human body fluids and belonging to the lipocalin superfamily. The monomer contains a single (183 amino acid residues long) peptide chain with 3 cysteine residues (2 of which form a disulfide bridge) and is glycosylated. The molecular mass of the glycosylated protein is about 27 kDa. Native gel electrophoresis results revealed partial oligomerisation of protein HC, which therefore was analysed by gel filtration. Two forms (monomer and dimer) of the protein HC were isolated. The SAXS data were recorded on an X33 camera using synchrotron radiation (lambda=0.15 nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg, Germany). Solution scattering results permitted determination of the structural parameters of both forms of the protein studied. The monomer of protein HC is characterised by a radius of gyration R(G)= 2.20 nm and D(max)=6.3 nm and the dimer by R(G)=2.99 nm and D(max)=9.5 nm.

Absence of increased alpha1-microglobulin in IgA nephropathy proteinuria.

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only alpha(1)-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of alpha(1)-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of alpha(1)-microglobulin forms a complex with IgA in serum. These results suggest that alpha(1)-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that alpha(1)-microglobulin might have a relationship with IgA nephropathy pathology.

Production of recombinant human alpha1-microglobulin and mutant forms involved in chromophore formation.

Alpha(1)-Microglobulin, a 26 kDa lipocalin present in plasma and tissues, carries a set of unknown chromophores, bound to C34, K92, K118 and K130, which cause its charge and size heterogeneity. In man, the protein is found in two forms, full length and lacking the C-terminal tetrapeptide LIPR (t-alpha(1)-microglobulin), both which are heme-binding and the latter with heme-degrading properties. We report cloning and overexpression of full length alpha(1)-microglobulin (wt protein), t-alpha(1)-microglobulin (wtdeltaLIPR) and the mutants C34S, K(92,118,130)T and C34S/K(92,118,130)T, the latter subsequently abbreviated as K(3)T and C34S/K(3)T, in Escherichia coli. After purification and refolding from inclusion bodies, all proteins were correctly folded as determined by far-UV circular dichroism and radioimmunoassay. As revealed by gel filtration, recombinant alpha(1)-microglobulins had lower tendencies to form dimers than human plasma or urine analogues. All alpha(1)-microglobulin forms displayed higher amounts of the chromophore than bovine serum albumin but significantly lower than the human urine or plasma counterparts. Differences in the absorbance and fluorescence profiles are consistent with a model where the chromophore is formed by a series of reactions with heme or other chromophore precursors and where C34 is essential for binding of the ligand, K92, K118 and K130 are involved in transformation into the chromophore and LIPR inhibits the latter reaction.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid.

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

Proteomic characterization of inter-alpha inhibitor proteins from human plasma.

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.