Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

Plasma levels of soluble VEGF receptor isoforms, circulating pterins and VEGF system SNPs as prognostic biomarkers in patients with acute coronary syndromes.

BACKGROUND: Development of collateral circulation in coronary artery disease is cardio-protective. A key process in forming new blood vessels is attraction to occluded arteries of monocytes with their subsequent activation as macrophages. In patients from a prospectively recruited post-acute coronary syndromes cohort we investigated the prognostic performance of three products of activated macrophages, soluble vascular endothelial growth factor (VEGF) receptors (sFlt-1 and sKDR) and pterins, alongside genetic variants in VEGF receptor genes, VEGFR-1 and VEGFR-2. METHODS: Baseline levels of sFlt-1 (VEGFR1), sKDR (VEGFR2) and pterins were measured in plasma samples from subgroups (n = 513; 211; 144, respectively) of the Coronary Disease Cohort Study (CDCS, n = 2067). DNA samples from the cohort were genotyped for polymorphisms from the VEGFR-1 gene SNPs (rs748252 n = 2027, rs9513070 n = 2048) and VEGFR-2 gene SNPs (rs2071559 n = 2050, rs2305948 n = 2066, rs1870377 n = 2042). RESULTS: At baseline, levels of sFlt-1 were significantly correlated with age, alcohol consumption, NTproBNP, BNP and other covariates relevant to cardiovascular pathophysiology. Total neopterin levels were associated with alcohol consumption at baseline. 7,8 dihydroneopterin was associated with BMI. The A allele of VEGFR-2 variant rs1870377 was associated with higher plasma sFlt-1 and lower levels of sKDR at baseline. Baseline plasma sFlt-1 was univariately associated with all cause mortality with (p < 0.001) and in a Cox's proportional hazards regression model sFlt-1 and pterins were both associated with mortality independent of established predictors (p < 0.027). CONCLUSIONS: sFlt-1 and pterins may have potential as prognostic biomarkers in acute coronary syndromes patients. Genetic markers from VEGF system genes warrant further investigation as markers of levels of VEGF system components in these patients. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry. ACTRN12605000431628 . 16 September 2005, Retrospectively registered.

Novel haemoglobin mutation (α127Lys→Glu) increases oxygen affinity and has a minor effect on haptoglobin binding.

OBJECTIVES: To determine if a new haemoglobin (Hb) variant was the underlying cause of erythrocytosis in a subject with a high apparent HbA(1)c. DESIGN AND METHOD: Haemolysate was analysed by ESI MS, and individual components purified by ion exchange and reverse phase chromatography. Peptide mapping was used to pinpoint the substitution and DNA sequencing to confirm the precise mutation. Oxygen affinity was measured and relative haptoglobin (Hp) binding estimated. RESULTS: Intact protein analysis and peptide mapping suggested a mutation in peptide α13 and DNA sequencing confirmed a novel α127Lys→Glu substitution in the α 2 gene. The abnormal Hb had a significantly higher O(2) affinity (5.8 mmHg) than HbA (12.4 mmHg). In addition the mutation caused a small but significant decrease in Hp binding. CONCLUSION: Molecular models show that the side chain of α127Lys stabilises the T structure of deoxy Hb and that mutation to Glu would favour conversion to the high affinity R state. Notwithstanding this and the demonstrated high affinity, there was only a small increase in RBCs, Hb concentration and PCV in other female carriers of the mutation. The absence of a significant phenotype of erythrocytosis is most probably due to the low level (19%) of the variant.

Novel hemoglobin alpha chain elongation resulting from a 15-residue insertion and tandem duplication of the F helix.

OBJECTIVES: To determine the cause of an unusual hemoglobin pattern with two novel components eluting after HbA(2) on cation exchange HPLC. This variant was detected during HbA1c measurement and was associated with a normal blood count and a positive isopropanol test. DESIGN AND METHOD: Whole hemolysate and isopropanol precipitates were analysed by ESI MS, and individual components were purified by reverse phase and cation exchange HPLC. Tryptic peptide mapping of isopropanol precipitates was used to detect the molecular lesion and DNA sequencing was used to characterise the precise rearrangement. RESULTS: ESI MS showed a mass increase of 1614Da in 9% of the alpha globin chains and sequence analysis of the alpha2 gene revealed the heterozygous insertion of 45 nucleotides after codon 93. The predicted in phase incretion of ALSALSDLHAHKLRV (+ 1613Da) is a direct repeat of residues alpha79-93 and signature ions from the new peptide were clearly visible in peptide maps of the unstable hemoglobin. CONCLUSION: The insertion probably results from replication slippage during DNA synthesis and the 15-residue repeat results in full repetition of the heme-linked F helix. The nature of the inserted sequence explains the molecular instability and the electrophoretic mobility, but not the twin peaks observed on cation exchange HPLC. These components had the same chain composition, (alpha(L) beta), the same number of heme groups per chain, were not in rapid equilibrium with each other, and probably represent hemoglobin species with different conformers of the elongated alpha(L) chain.

Convenient high-resolution isoelectric focusing (IEF) method for the separation of alpha1-proteinase inhibitor (A1PI) isoforms in A1PI concentrates.

Currently, high-resolution separation of A1PI is done in highly specialized laboratories using gels made in-house. This paper presents a high-resolution method for the separation of A1PI concentrates and human plasma using commercially available gels. Hybrid IEF was performed with carrier ampholytes and the gels were stained with Coomassie Brilliant Blue G-250. In addition, a sensitive immunoblotting procedure is described. The IEF method allowed the reproducible and convenient determination of the IEF pattern of A1PI in concentrates including resolution of glycan-dependent isoforms and isoproteins with secondary modifications such a C-terminal Lys-truncation. Furthermore, a shift in the IEF pattern of A1PI occurring upon reduction could be detected. Finally, in combination with a sample pretreatment step, the method proved able to monitor complex A1PI isoform patterns in samples with low A1PI concentrations as present for example in bronchoalveolar lavage solutions.