The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicate that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A reduces readily in the presence of dithionite. Raman frequencies of the FeCO distortions in WT, H136A, and Y235A HmuT–CO complexes provide further evidence for the axial ligand assignments. Additionally, the se frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen-bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen-bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

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ObjectivesPoint of care testing (POCT) contributes to diagnosis and monitoring with fast testing time and easily performed assays. We evaluated the Abaxis Piccolo Xpress point of care chemistry analyzer using the Liver Panel Plus discs for our pediatric patient population at Texas Children's Hospital.Design and methodsAnalytical performance was evaluated for precision and linearity using quality control materials and commercially available verification samples. Comparison studies were performed between Piccolo Xpress analyzer and Vitros 5600 analyzer using patient samples. Interference studies were carried out using nine different patient pool sera. Lipemia interference was removed using LipoClear for severely lipemic sample pools.ResultsPrecision of all tests was excellent (CVs<5% for all measured analytes except TBIL). All assays were linear and accurate within the allowable total error. Comparison studies showed that three analytes (amylase, GGT and TBIL) had statistically significant bias. Interference study results did not exceed the total allowable error for hemoglobin (<150 mg/dL), bilirubin (<15 mg/dL) and lipemia (<400 mg/dL except ALT, GGT and TP). LipoClear treatment removed lipemia interference for all analytes except total protein.ConclusionsThe Piccolo Xpress chemistry analyzer showed an acceptable analytical performance for precision, linearity and interference from common substances. Increased bias for three analytes in comparison studies could be due to different methodologies.