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Structural Properties of Human Urine‐Sourced Neutrophil Gelatinase Associated Lipocalin (huNGAL)
Author(s) -
Rae Tracey D,
Bonn Ryan,
Workman Ryan,
Ali Salman,
Syed Hina,
Rupprecht Kevin,
Tu Bailin,
Fishpaugh Jeffrey,
Grenier Frank
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.863.10
Subject(s) - lipocalin , urine , immunoassay , chemistry , analyte , recombinant dna , blot , gelatinase , chromatography , microbiology and biotechnology , biochemistry , biology , enzyme , immunology , antibody , gene
Elevated levels of Neutrophil Gelatinase Associated Lipocalin (NGAL) in the urine have been established as a promising diagnostic indicator for acute kidney injury (AKI). Development of a quantitative diagnostic immunoassay for NGAL in urine prompts an understanding of the native protein analyte and its structural similarity to the selected recombinant protein used to calibrate the assay. Here we present findings from various analyses on the native human NGAL protein in urine (huNGAL). Chromatographic size‐fractionation of individual human urine specimens indicate that the predominant form of huNGAL is monomeric, although oligomeric forms are detected in some specimens. When observed, NGAL‐containing oligomers are shown to occur through inter‐protein disulfide linkage. An enriched huNGAL protein sample from a pool of urine specimens was analyzed by two‐dimensional electrophoresis (2DE) with Western Blotting to elucidate size and charge distribution of NGAL‐active isoforms. Although all identified huNGAL isoforms displayed molecular weights near the predicted intact polypeptide sequence, the charge distribution of the isoforms was surprisingly wide (pI 5.9‐9.1) given a theoretical pI of 9.0 for the native polypeptide. Structural properties of the enriched huNGAL are compared to those of the immunoassay calibrator protein as well as NGAL protein from other recombinant and native sources.