Per-Ingvar Ohlsson

The isolation and some liganding properties of lactoperoxidase

Lactoperoxidase is an animal protein which participates in antimicrobial mechanisms [I]. Its electron donor profile differs from that of plant peroxidases regarding halide ions [2]. Isolation procedures are available but somewhat tedious [3--S]. Plant isoperoxidases with different affinities for aromatic substrates can be separated on phenylSepharoseR [6]. This observation has now been developed into an isolation procedure for LP. Its essential features are alternations between column materials with ionic and hydrophobic binding forces. The opposite requirements for ionic strength minimize the number of dialyses. The binding of LP to phenyland octyl-Sepharose is compared to the binding of horseradish peroxidase to both Sepharoses. Attempts are made to relate these adsorptions to optically operable equilibria between the peroxidases and free, aromatic ligands. Plant peroxidases can be isolated by means of affinity chromatography on hydroxamic acid-BioGel AR [7]. An imidazolecarrying polysaccharide binds hemoglobin and myoglobin specifically and unspecifically [8]. Sepharose-concanavalin A binds the glycoprotein HRP [9,10].

A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.

Plasminogen activator inhibitor type 2 (PAI‐2) is the only wild‐type serpin that polymerizes spontaneously under physiological conditions. We show that PAI‐2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD‐loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis‐ANS to hydrophobic cavities indicate that the C79–C161 disulfide bond stabilizes PAI‐2 in a polymerogenic conformation with an open A‐β‐sheet. Elimination of this disulfide bond causes A‐β‐sheet closure and abrogates the polymerization. The finding that cytosolic PAI‐2 is mostly monomeric, whereas PAI‐2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI‐2 polymerization. Taken together, our data suggest that the CD‐loop functions as a redox‐sensitive switch that converts PAI‐2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

Optical, NMR and EPR properties of horseradish peroxidase and its donor complexes.

Complexes of horseradish peroxidase (HRP) and hydrogen donors have only recently been demonstrated spectrophotometrically [ 1,2]. The donor increased the dissociation of the HRl-cyanide complex and altered the optical spectrum of HRIHzOz compound II much more than that of free HRP, but no formation of an active ternary HRIHzOz -donor complex was assumed [ 11. EPR spectra of HRP without peroxide or donor (‘free’ enzyme) [2--63 have been variously interpreted as corresponding to high spin [2-41 or thermally mixed high and low spin species [6]. A transition from a multispecies EPR spectrum for the free enzyme to a pure high spin spectrum has been observed upon the addition of benzhydroxamic acid [2], but no EPR or NMR studies of HRI-donor complexes have been published. The present paper gives some optical, NMR, and EPR properties of HRP and its donor complex.** A recent analysis of the bacterial cytochrome c’ EPR spectrum is applied to the EPR spectrum of HRP [7,8].

Infrared spectroscopic evidence of hydrogen bonding between carbon monoxide and protein in carbonylhorseradish peroxidase C

Carbonylhorseradish peroxidase isoenzyme C2 (EC 1.11.1.7) exhibits two bands in the infrared spectrum attributable to the ligand CO at 1933.5 and 1905 cm−1. Replacement of H2O by D2O results in shifts to both bands to new positions at 1932.5 and 1902.5 cm−1. The results indicate strong hydrogen bonding to the terminal oxygen of CO, of strength comparable to that recently observed for oxyhemoglobin and oxymyoglobin.

Heart failure caused by a novel amyloidogenic mutation of the transthyretin gene: ATTR Ala45Ser

Cardiac failure in transthyretin (TTR) amyloidosis patients has been shown to be caused by different mutations in the TTR gene. In the present case, a 73-year-old man from Northern Sweden was evaluated for heart failure. Amyloid deposits were found in subcutaneous fat and in intestinal biopsies. The presence of a variant form of TTR was detected in the plasma by electrospray ionisation mass spectrometry (ESI-MS). The mutation was located by single-strand conformation polymorphism (SSCP) analysis of the TTR gene where a band shift was seen in exon 2. Direct sequencing of exon 2 revealed a single base-pair substitution (G1724T). This transversion results in an amino acid substitution at codon 45, alanine to serine (ATTRAla45Ser). Mass spectrometry analysis excluded that the variant is a polymorphism, since no similar shift in molecular weight has been present in more than 200 control samples. Congo red and immunostaining of duodenum biopsy specimens confirmed the presence of systemic ATTR amyloidosis, and clinical examination, including echocardiography, found evidence of a restrictive cardiomyopathy. He had 10 years previously been operated for a bilateral carpal tunnel syndrome, but otherwise no symptoms were present that could be attributed to his systemic amyloidosis. No axonal polyneuropathy was noted at nerve conduction studies. This novel mutation is the second amyloidogenic TTR mutation found in the Swedish population.

Rapid screening for amyloid-related variant forms of transthyretin is possible by electrospray ionization mass spectrometry

We have used a new and rapid method to detect three variant forms of transthyretin (TTR): methionine for valine at position 30 (Met‐30), serine for cysteine at position 6 (Ser‐6) and methionine for leucine at position 111 (Met‐111). By using an anti‐transthyretin antibody and a centrifugal concentrator, transthyretin was isolated from plasma samples and analysed for variant forms by electrospray ionization mass spectrometry. Differentiation between homozygous and heterozygous transthyretin Met‐30 and Met‐111 posed no problems. However, a clear separation of transthyretin Ser‐6 and Met‐111 peaks in one patient with concordant Ser‐6 and Met‐111 mutations could not be achieved. The present method enables a quick and reliable detection of variant TTR. It can be used to screen families or small populations for abnormal TTR. Knowledge of TTR polymorphism and the variable expression of different amyloidogenic mutations should be taken into consideration when applying the methods in clinical practice.

Liver transplantation in transthyretin familial amyloid polyneuropathy: first report from Argentina.

This is the first report from Argentina of liver transplantation in patients with transthyretin related familial amyloidotic polyneuropathy. The aims of the study were to analyze the clinical characteristics of this new focus and evaluate the postoperative complications and long term follow up. Five of ten patients evaluated underwent liver transplantation. During the waiting period the polyneuropathy disability score in each patient progressed one or two stages. Pretransplant modified body mass index was 723. The procedure was done with full size grafts in four cases and a split right graft in one. All patients presented postoperative complications related to disease: severe edema of the legs, recurrent choledochal lithiasis, postoperative hernia, necrotizing fasciitis and ischemic rectosigmoidal perforation. Assessment of three patients after 20 months of transplantation showed improvement in somatic and mental symptoms. No improvement was noted in cardiac denervation and gastric stasis. Liver transplantation is a rational therapeutic option for transthyretin familial amyloidotic polyneuropathy in Argentina and should be indicated in earlier stages of the symptomatic disease to reduce the postoperative morbidity and mortality. Family studies and follow up of asymptomatic carriers will define the epidemiological behavior in this country and facilitate early therapeutic intervention.

THE BARRIER FOR HEME-PROTEIN SEPARATION ESTIMATED BY NON-EQUILIBRIUM MOLECULAR DYNAMICS SIMULATIONS

Abstract In heme-containing proteins the heme group is usually non-covalently bound in a pocket. Molecular dynamics (MD) simulations have been performed to estimate the barrier height for heme–protein separation. In simulations of myoglobin dissolved in water, a force has been applied to pull the heme out of the binding pocket. With forces above 0.5 nN, the heme group is easily pulled out of the pocket in times of the order of tens of picoseconds. With weaker forces, heme release becomes too slow to be monitored in an MD simulation covering a couple of hundred picoseconds. These results are consistent with a free energy barrier to heme release of about 100 kJ/mol. The results show that the main energetic change that occurs during the release is a conversion of heme/protein Lennard–Jones energy into heme/water Lennard–Jones energy. The release is essentially barrierless in energy indicating that the main part of the barrier is entropic.

THE SPONTANEOUS HEMIN RELEASE FROM LUMBRICUS TERRESTRIS HEMOGLOBIN

The slow, spontaneous release of hemin from earthworm, Lumbricus terrestris, hemoglobin has been studied under mild conditions in the presence of excess apomyoglobin. This important protein is surprisingly unstable. The reaction is best described as hemin released from the globin into water, followed by quick engulfment by apomyoglobin. The energetics of this reaction are compared with those of other types of hemoglobins. Anomalously low activation energy and enthalpy are counterbalanced by a negative entropy. These values reflect significant low frequency protein motion and dynamics of earthworm hemoglobin and may also indicate an open structure distal to the heme. This is also supported by the infrared spectrum of the carbonyl hemoprotein, which indicates several types of distal interactions with the bound CO. The reported low heme to polypeptide ratio for this protein may be due to facile heme and hemin release by the circulating protein.

Temperature dependence of CO ligation to LegHb and Mb: 0-80°C

Abstract The temperature dependence of the coordination of CO to two hemoproteins, myoglobin (Mb) and leghemoglobin (LegHb), has been studied by absorption spectroscopy. All spectral changes in the electronic and infrared ranges are fully reversible between 8 and 88°C and indicate a more linear orientation of the distal ligand with increasing temperature. The characteristic energy for the above spectral changes is around 200 cm −1 or identical to the Fe-Imidazole (Im) mode in hemoproteins. Our results are also compared with recent models for the dynamics of CO adsorbed at the surfaces of transition metals. Based on these surface models we suggest that our observed temperature dependences stem from thermal excitations of the Fe-Im vibration. This implies a so far undocumented mechanism by which the Fe-proximal bond influences the binding and energy dissipation at the active site of hemoproteins.