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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.

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.

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.

Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I(-) into antimicrobials and small aromatic organics after being itself oxidized by H(2)O(2). LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO-LPO at 2.0Å resolution and infrared (IR) spectra of the iron-bound CO stretch from pH 3 to 8.8 at 1 cm(-1) resolution. The observed Fe-C-O bond angle of 132° is more acute than the electronically related Fe(III), CN-LPO with a Fe-C-N angle of 161°. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III) moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN-LPO. The IR spectra of CO-LPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ≈ 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH 7; both band widths at half-height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.

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.

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-beta-sheet. Elimination of this disulfide bond causes A-beta-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.

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.

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.

Slow heme transfer from horseradish peroxidases C2 and A2, cytochrome c peroxidase, chloroperoxidase, and leghemoglobins to a heme acceptor protein, apomyoglobin, has been studied under mild conditions. The reaction is best described as heme release into water followed by quick engulfment by apomyoglobin. The energetics of the activated process are large and interpreted as connected to both polypeptide motions during release and the ordering of water around the heme during solvation. The free energy required to break the iron(III)-ligand 5 (L5) bond is a minor but crucial portion of the activation free energy. Donor-acceptor protein interactions are not involved in the transfer. Fast heme release from inactive protein has also been observed. Apoprotein recombination with porphyrins and hemes suggest that this lack of activity is a result of Fe-L5 bond breaking. Slow heme transfer from horseradish peroxidases C2 and A2, cytochrome c peroxidase, chloroperoxidase, and leghemoglobins to a heme acceptor protein, apomyoglobin, has been studied under mild conditions. The reaction is best described as heme release into water followed by quick engulfment by apomyoglobin. The energetics of the activated process are large and interpreted as connected to both polypeptide motions during release and the ordering of water around the heme during solvation. The free energy required to break the iron(III)-ligand 5 (L5) bond is a minor but crucial portion of the activation free energy. Donor-acceptor protein interactions are not involved in the transfer. Fast heme release from inactive protein has also been observed. Apoprotein recombination with porphyrins and hemes suggest that this lack of activity is a result of Fe-L5 bond breaking. Upprättat; 1991; 20080613 (ysko)

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.

Dicarboxidine is a low- or noncarcinogenic benzidine-type chromogenic substrate to peroxidases. The color formation is of zero order from the onset of the reaction with a rate proportional to the enzyme concentration. Substrate solution and absorbance are stable for at least 24 h, the latter after an early decrease of 4% in 10 min. The complexity of the benzidine reaction necessitates precautions against side reactions. These are analyzed and suitable assay conditions are presented. Dicarboxidine is a valuable chromogen for studies of a slow generation of hydrogen peroxide, a slowly reacting substituted hydroperoxide, or a low peroxidase activity such as in the enzyme-linked immunosorbent assay or other immunological tests.

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.

Correlations between reduction potential, CO stretch frequency and CO half-band width in hemeproteins

The dithionite ion is catalytically disproportionated by lactoperoxidase with Km = 0.36 mM in 100 mM glycine HCl pH 3.0. The products formed are thiosulfate and hydrogensulfite ions. The rate of reaction is considerably increased at low pH with a pKa at 3-3.5 possibly indicating the involvement of a carboxyl group. The reaction is competitively inhibited by hydrogensulfite, Ki = 5.5 mM in 100 mM glycine HCl pH 3.50. Four different spectral forms of reduced lactoperoxidase appear during the reaction. The first two forms are found during the lag phase of the reaction. The third form, which is interpreted as a ternary complex, exists under the dismutation phase. After exhaustion of the substrate a visible spectrum similar to that of lactoperoxidase H2O2 compound III appears. A mechanistic model for the lactoperoxidase dismutation of the dithionite ion is proposed and discussed.

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].

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.

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.

A multitude of external signals induce extensive phosphorylation of Oncoprotein 18 (Op18), which suggests a putative role for this protein in signal transduction. We have recently identified two distinct proline-directed kinase families that phosphorylates Op18 with overlapping but distinct site preference. These two kinase families, mitogen-activated protein (MAP) kinases and cyclin-dependent cdc2 kinases, are involved in receptor- and cell cycle-regulated phosphorylation events, respectively. In the present study, site-specific phosphorylation of Op18 in response to stimulation of the antigen receptor-associated CD3 complex was analyzed in the Jurkat T cell-line. The results show that CD3-induced phosphorylation of Ser-25 of Op18, which is the primary MAP kinase phosphorylation site, can be induced by an apparently protein kinase C (PKC)-independent signal transduction pathway. We also demonstrate that Ser-16 of Op18 is specifically phosphorylated in response to the Ca2+ signal generated by CD3 stimulation or by the Ca2+ ionophore ionomycin. Ser-16 phosphorylation occurs independently of both PKC and MAP kinase activation. Using site-specific Op18 mutants and tryptic phosphopeptide mapping, we show that phosphorylation of Ser-16 of Op18 together with Ser-25, or Ser-25 and Ser-38, generates two Op18 phosphoisomers showing a dramatic electrophoretic retardation. In conclusion, site-mapping studies of Op18 reveal that CD3 stimulation results in an apparently PKC-independent activation of both the MAP kinase and a Ca(2+)-regulated kinase pathway, which results in phosphorylation of distinct sites of Op18. The data also pinpoints the specific phosphorylation events that result in electrophoretic retardation of Op18.

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.

Lactoperoxidase (LP) [1] is a remarkable enzyme, being an efficient scavenger of peroxides, an oxidant of usual peroxidase substrates and halide ions [2] while also catalyzing the disproportionation of dithionite [3]. Like other peroxidases LP has a low redox potential and a protoheme prosthetic group, but a mixed spin type spectrum and a strong heme–protein attachment single out LP as being unique. For this reason we have studied the CO stretch of LP · CO at pH 5–9. The CO stretch of carbonyl horseradish peroxidases (HRP) A and C is pH dependent in terms of number and positions of ir bands [4]. This was interpreted as indicating the presence of a titratable group near the active center influencing the carbonyl resonator at low pH by H-bonding to the oxygen atom in CO. The ir spectrum of LP · CO exhibits CO stretches at 1940, 1955, and 1961 cm− (Fig.1 Spectra at intermediate pH values omitted). The 1940 cm− band predominates, and its position is independent of pH and buffer species. The 1955 cm− band is intense at ph 5, gives a shoulder at pH 7, and has vanished at pH 9. The 1961 cm− band changes in the reverse manner. The low intensity and essentially pH-independent band at ∼1913 cm− may not be related to bound CO. The ir spectra of LP and HRP A, C have in common a) a pH-independent stretch at 1937 ± 3 cm− with a half-band width 12 cm−; b) a broader band, sensitive to protons or dipoles. Stretch a) is attributed to CO in a narrow, apolar environment, whereas in b) CO may dwell in a more open site. The balance between the 1955 and 1961 cm− stretches may depend upon a change in polarity due to a proton or, more likely, a polar group. The properties a) and b) seem to be unique to peroxidases, although at present there is no explanation of why stretch b) is located on opposite sides of a) in HRP and LP. The active site of lactoperoxidase may be different from that of other peroxidases, either lacking the distal base, or the ligand binding sites being rigid and preventing carbonyl interaction with the distal base [5]. The redox potentials of LP and HRP A, C (−180, −212, and −265 mV) and vCO (1940, 1938, and 1933 cm−) of the a) stretch follow the correlation found for other heme proteins [4].