The CRP preparation contained two very faint bands on either side

The CRP preparation contained two very faint bands on either side of the 94 kD marker, in addition to CRP (Fig. 1b). These other proteins comprised less than 1% of the total protein and their presence is not surprising because several chromatographic steps required to remove traces of other proteins

in preparing the most highly purified CRP (de Beer and Pepys, 1982, Hawkins et al., 1991 and Carlucci http://www.selleckchem.com/products/Roscovitine.html et al., 2010) were not possible in the present pharmaceutical GMP procedure. The trace higher molecular weight proteins were identified by proteomic analysis, using mass spectrometry of trypsin digested fragments of the bands excised from the SDS‐PAGE. The lower mass band was the μ heavy chain of IgM and the higher mass band was plasmin/plasminogen (data not shown). The very faint trace bands

of mass lower than the SAP and CRP protomers are characteristic cleaved fragments of the protomers which are derived from intact pentameric pentraxins when they are reduced and denatured; they are invariably Talazoparib ic50 present in pentraxins isolated from ex vivo human material. No SAP was detected in the CRP preparation and no CRP was detected in the SAP preparation, which were tested at 3.0 and 1.5 mg/mL respectively using assays which in both cases detected the other pentraxin at 1 μg/mL ( Nelson et al., 1991 and Shine et al., 1981). The authentic covalent structures of the protomers of each pentraxin were confirmed by ESIMS, with average molecular masses (SD) (n = 3) of 25,462.64 (0.39) for SAP and 23,027.46 (0.52) for CRP, corresponding exactly to the predicted masses for their respective amino acid sequences, plus glycan in the case of 3-oxoacyl-(acyl-carrier-protein) reductase SAP (Pepys et al., 1994) and with N‐terminal PCA in CRP ( Oliveira et al., 1979). Integrity of the authentic native non‐covalent pentameric assembly of each protein was confirmed by gel filtration chromatography, which also showed the absence of any aggregation or dissociation into free protomers ( Fig. 2). The same result was obtained with the SAP preparation in non‐denatured gradient PAGE ( Fig. 3). Unlike the 4-30% gradient gels in Tris glycine we have previously used (

de Beer et al., 1982) but which are no longer available, human CRP does not form a discrete band in the present system. Functional activity of the proteins was confirmed by their reproducible, 100%, strictly calcium dependent binding to phosphoethanolamine-Sepharose beads (not shown). Furthermore these human proteins had the expected plasma clearance half life of ~ 3-4 h after intravenous injection into normal wild type C57BL/6 mice (Baltz et al., 1985 and Hawkins et al., 1988a) (shown for SAP in Fig. 4; not shown for CRP). This is a very sensitive test for structural and functional integrity of plasma proteins as even extremely subtle alterations, which may be undetectable by in vitro biophysical and biochemical methods, cause accelerated clearance of plasma proteins from the circulation in vivo.

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