This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The cardiac hormone B-type natriuretic peptide (BNP) is synthesized in myocytes as a prepro 134-amino acid residue molecule. The 108-residue proBNP mature form of the hormone is proteolytically cleaved to a biologically active form of 32 amino acids (residues 77108) and an N-terminal fragment (residues 176; NT-proBNP) with an as yet undefined biological function (1). Clinically, both BNP and NT-proBNP have shown great promise as secreted, bloodborne diagnostic markers of left ventricle dysfunction. Measurement of each is based on immunoassays; it therefore is likely that changes in the molecular form, e.g., posttranslational modifications, further proteolytic processing, and an oligomeric state for either analyte, could affect their measurements (2)(3)(4)(5). These types of confounding molecular issues are likely for many analytes, with the myocyte damage marker cardiac troponin I one of the better studied. In this case, the commercial assays use antibodies directed to different epitopes, making 'universal' calibration and determination of absolute analyte concentration difficult (6). A previous report has indicated that NT-proBNP exists as a coiled-coil trimer, based on size-exclusion HPLC (SE-HPLC) of human, plasma-extracted material and a computer algorithm that predicts coiled-coils (7). I reinvestigated this claim on synthetic NT-proBNP, using the physicochemical techniques of analytical sedimentation, equilibrium ultracentrifugation, and circular dichroism (CD), and demonstrate that NT-proBNP is a monomer and not a trimer. NT-proBNP was produced by solid-phase peptide synthesis (AnaSpec, Inc.) and obtained as a gift from Dade-Behring (Newark, DE). I used N-terminal sequencing and mass spectrometry as quality assurance procedures. Edman sequencing (8) was performed by Midwest Analytical, Inc. on 2 Coomassie-stained Immobilon-PSQ (Sigma) NT-proBNPcontaining membrane sections. The first 52 residues were positively identified, with no preview, before the signal was not discernable from background (data not shown).
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