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Hemeproteins can act as catalysts, oxygen carriers or electron conductors. The ferric/ferrous reduction potential E(m7) of iron in the center of the prosthetic group ranges from negative values for peroxidases to an extreme positive value for cytochrome a(3) with Hb and Mb in the middle [1]. Proteins exercise their influence on E(m7) in several ways: via substituents at the periphery of the chelate structure, via the proximal ligand, and via interaction with the surrounding medium, amino acid side chains, or polar solvents. Work on recombined proteins and 2,4-substituted free hemes documented that the first two effects are additive [2]. For the third effect, models of the dielectric media on a molecular level have been successfully applied [3-5]. E(m7) has also been empirically correlated to the degree of heme exposure to water [6-8]. The apoprotein/porphyrin and water/porphyrin interfaces are complementary since water molecules fill any empty space in the crevice and surround any pertinent part of heme outside the protein boundary. The present work links to this idea by a combination of statistical mechanics simulations and quantum mechanical calculations comparing heme in water with heme in an apolar environment. Our results show that polarization of the porphyrin pi-electron cloud by the field from water dipoles influences E(m7). The dominant effect of this and other determinates of iron electron availability is perturbations of delocalized electron density in the porphyrin chelate, reproduced by a model where the prosthetic group is treated as a disc of uniform electron density. The present work is also of interest since the interfacial energy constitutes the main barrier for heme-protein separation [9-11].

Background For all forms of amyloidosis, the amyloid-generating mechanism is unknown. Familial amyloidotic polyneuropathy type I is caused by a variant transthyretin (TTR Met-30). As electrospray ionization mass spectrometry (ESI-MS) discloses both thiol-conjugated and -unconjugated forms of wild-type and variant TTR, we wanted to investigate the relationship between TTR conjugation and clinically overt amyloid disease. Methods Plasma from 35 individuals (12 symptomatic TTR Met-30 carriers, nine asymptomatic and 14 healthy control subjects) were analysed using ESI-MS. Results The total TTR concentration was significantly lower in symptomatic TTR Met-30 carriers than in control subjects. An increased percentage of conjugated TTR Met-30 was found in symptomatic carriers compared with asymptomatic, whereas the percentage conjugated wild-type TTR was similar for control subjects, asymptomatic and symptomatic TTR Met-30 carriers. Conclusion The finding of a decreased ratio of unconjugated to conjugated TTR Met-30 in plasma samples from symptomatic TTR Met-30 carriers indicates that thiol conjugation of TTR could be involved in amyloid formation.

Structural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins.

Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

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 (ATTR Ala45Ser). 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.

Physiological fibrinolysis is a proteolytic degradation of polymerized fibrin. A particular fibrinolytic enzyme system is present in blood. The central reaction in this system is the activation of a proenzyme, plasminogen, to the proteolytic enzyme plasmin. The activation is triggered by highly specialized proteases, plasminogen activators. One of these, the tissue plasminogen activator (tPA), is synthesized by endothelial cells and excreted to the blood on certain stimuli. Properties, which distinguish tPA from other types of plasminogen activators are a high affinity to fibrin and the very strong stimulation exerted by fibrin on tPA induced activation (2–3 orders of magnitude). These properties have evoked the idea of an efficient ternary activation complex between tPA, plasminogen and fibrin. The work on the isolation of tPA, the determination of its primary structure and gene structure as well as elucidation of its physicochemical properties has been performed by a large number of scientists. Several reviews have been published (e.g. Collen 1980; Ranby and Wallen 1985; Dano et al. 1985; Erickson et al. 1985; Wallen 1987). Fig. 1 shows the now well established primary structure* of wild tPA.

The reduction of lactoperoxidase with sodium dithionite has been studied by means of stopped-flow spectrophotometry in an anaerobic system. Under pseudo-first-order conditions the rate constant was found to be linearly dependent on the square root of the dithionite concentration, which confirms the monomeric radical, SO2- as the reducing species. The second-order rate constant is moderately influenced by increased ionic strength but drastically increased at lower pH. The pH dependence supports the previously suggested existence of a carboxyl group, essential to the different enzymatic functions of lactoperoxidase. The second-order rate constant for the reduction of lactoperoxidase at pH 7.0 (kappa 1 = 1.3 X 10(5) M-1 s-1) was about three times higher than the rate constant for the reduction of cyanide-bound lactoperoxidase and two times the rate constant for the reduction of the fluoride-lactoperoxidase complex.

Two properties of lactoperoxidase (LP, E.C.1.11.1.7) are of biological interest, its catalytic activity and its stickyness. It is very active as a peroxidase, acts as an oxygen transferase, and dismutates hydrogenperoxide and dithionite. The protein structure is rigid and the heme group seems to be well hidden. LP adheres firmly to various kinds of surfaces such as tooth enamel, glass, plastics, or octyl-Sepharose®.

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.

We have developed a quick and reliable diagnostic method for detecting variant forms of transthyretin (TTR); namely, centrifugal concentration followed by electrospray ionization mass spectrometry (ESI-MS). Argentinian patients from three families with neuropathic amyloidosis and their relatives were screened for mutated TTR by ESI-MS. In order to facilitate transportation, we investigated the impact storage had on lyophilized anti-TTR-antibody precipitates' mass spectra. For this investigation, plasma samples from three Swedish patients with known TTR amyloidosis were analysed. We detected identical, additional peaks corresponding to a variant form of TTR in 10 members of the families, and also in a lyophilized sample sent unfrozen by mail from Argentina. All except one symptomatic subject had additional peaks, the exception having undergone a liver transplantation for the disease. All patients were early onset cases, i.e. below 35 years of age, and family history suggests an aggressive, rapidly progressing disease. Lyophilized anti-TTR-antibody precipitates stored at room temperature for 1 week exhibited only minor differences compared with plasma samples stored at -70 degrees C. In a new Argentinian study on familial amyloidotic polyneuropathy, the variant TTR was quickly identified and typed by ESI-MS. To facilitate transportation, dry-frozen samples can be used and the quality of the spectra is similar to that of samples stored at -70 degrees C.

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.