The iron of lactoperoxidase is predominantly high-spin at ambient temperature. Optical spectra of lactoperoxidase indicate that the iron changes from high-spin to low-spin in the temperature range from room temperature to 20 K. The transformation is independent of whether the enzyme is in glycerol/water or solid sugar glass. Addition of the inhibitor benzohydroxamic acid increases the amount of the low-spin form, and again the transformation is independent of whether the protein is in an aqueous solution or a nearly anhydrous sugar. In contrast to lactoperoxidase, horseradish peroxidase remains high-spin over the temperature excursion in both solvents and with addition of benzohydroxamic acid. We conclude that details of the heme pocket of lactoperoxidase allow ligation changes with temperature that are dependent upon the apoprotein but independent of solvent fluctuations. At low pH, lactoperoxidase shows a solvent-dependent transition; the high-spin form is predominant in anhydrous sugar glass, but in the presence of water, the low-spin form is also present in abundance. The active site of lactoperoxidase is not as tightly constrained at low pH as at neutrality, though the enzyme is active over a wide pH range.

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.

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].

Two patients with amyloidosis caused by transthyretin (TTR) were investigated by immunohistopathologic, mass spectrometric, and molecular genetic methods. After confirming the immunoreactivity of TTR in the amyloid deposits using anti-TTR polyclonal antibody, a new method: centrifugal concentration and electrospray ionization mass spectrometry (ESI-MS) was employed to detect the variant TTR in the serum. Only 50 μl of the serum and 30 μl of the anti-TTR antibody were needed for the analysis. After incubation with the antibody, the samples were passed through a 1000 kDa cut off centrifugal concentrator to retain the antibody, thereafter, the filtrate was analyzed by ESI-MS. Several forms of normal and variant TTR were detected in the serum samples: unconjugated TTR, cysteine and cysteine–glycine conjugated TTR. In the patients, a variant form of TTR was detected with a 26.0 Da higher molecular weight than that of normal TTR. Single-strand conformation polymorphism (SSCP) and direct sequence analysis confirmed the presence of a one-base substitution situated at the codon 50 from AGT (Ser) to ATT (Ile) in both patients, that corresponded to the increased molecular weight of 26.0. The present diagnostic procedure demonstrates the usefulness of both ESI-MS and SSCP to screen for TTR related amyloidosis rapidly. Moreover, the DNA samples obtained from the band showing abnormal electrophoretic migration pattern in SSCP, facilitate the direct sequence analysis to detect the unknown mutation, and the observed shift in molecular weight of the variant TTR in ESI-MS confirms the base substitution.

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and Non-Isotopic RNase Cleavage Assay (NIRCA) respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12 Da higher molecular weight than normal TTR. NIRCA analysis suggested that the mutation exists near either the 5' or 3' end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5' end of exon 3. The molecular weight shift of this mutation was 12 Da, consistent with the result of ESI-MS.

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.

Variant forms and post-translational modifications of transthyretin (TTR) can be identified by electrospray ionisation mass spectrometry (ESI-MS). The aim of the present study was to investigate thiol conjugation of transthyretin and it's relation to age and symptomatic amyloid disease in different populations of variant TTR carriers. Plasma samples from 70 individuals from Denmark, Argentina, Sweden and Japan, with 2 different TTR mutations were analysed. The percentage cysteine (Cys) conjugated wild and variant TTR were calculated from the corresponding peaks of the spectra, and multiple regression analysis was employed to disclose relationships between age, symptomatic amyloid disease and origin. Age, origin and presence of symptomatic disease, were found to be independent factors related to transthyretin conjugation. A higher percentage of conjugated to unconjugated TTR was disclosed in symptomatic, but not in asymptomatic carriers. In summary: Thiol conjugation of TTR is dependent on age and presence of symptomatic amyloid disease. Furthermore, it varies between different populations. Variant TTR is more susceptible to thiol conjugation than the wild type. Post-translational factors may be related to amyloid formation and/or toxicity.

The aim of the present study was to analyze the forms of wild type and mutated monomeric transthyretin (Val30Met) in the amyloid fibrils of patients with familial amyloidotic polyneuropathy by electrospray ionization mass spectrometry (ESI-MS). The solubility of amyloid fibrils from the vitrectomized samples was examined to determine the appropriate solution for ESI-MS. ESI-MS analysis revealed that heterozygotic Val30Met amyloid fibrils contained 14.6 +/- 7.5% normal TTR. In all samples, 3 different types of variant ATTR could be identified: Full length ATTR, and -57, and -157 (or 156) Da from ATTR Val30Met were found. The two peaks showing -57, and -157 (or 156) Da from ATTR Val30Met corresponded to the -Gly, and -Gly-Pro sequences of ATTR Val30Met from the N-terminal. The results illustrate the heterogeneity of ATTR amyloid deposits and this method may be very useful for analyzing amyloid fibrils in ATTR related amyloidosis.

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5′ or 3′ end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5′ end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.

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 performed biochemical and immunological examinations of heterozygotic carriers of the transthyretin (TTR) mutant Y114C associated with familial amyloidotic polyneuropathy (FAP). The total serum TTR levels in Y114C TTR carriers were extremely low when analyzed by single radial immunodiffusion (SRID), whereas by indirect enzyme-linked immunosorbent assay (ELISA) procedure, their total TTR concentrations were increased. Recombinant homozygotic Y114C TTR showed no immunoreactivity towards a TTR antibody when analyzed by SRID, whereas by the ELISA procedure presented the same degree of reactivity as that of normal TTR or isolated serum heterozygotic Y114C TTR. These results indicate that immunodifusion based techniques cannot properly determine TTR serum levels in Y114C carriers. Analyses of serum TTR of the Y114C TTR carriers by electrospray ionization mass spectrometry (ESI-MS) with the orifice corn voltage at 60 V revealed a small peak of the free Y114C TTR in addition to large TTR peaks of normal TTR. The levels of the free mutant TTR increased with the orifice corn voltage at 90 V. In contrast, increase in orifice voltage from 60 to 90 V produced a reduction in the level of normal TTR. The results suggest a different pattern of association between monomers in Y114C relative to normal TTR.

Electronic changes of iron-and metal free porphyrins are reviewed in light of their importance to modify the ferri/ferro reduction potential and energy dissipation between ligated carbon monoxide and the porphyrin chelate. Substitutions at positions 2 and 4 are effective ways to exercise this influence. Other measures include exposure to water and modifications of axial ligation, including the 'three-ligand' case. These measures will also influence the stability of recombined hemin-protein entities, as apparent from the kinetics of heme-transfer from a modified donor protein to apomyoglobin, but the stability appears to be related to the bulkiness of 2,4-substitutional groups rather than to electron availability. It is suggested that the altered stability is a secondary effect from enhanced exposure to water in the crevice.

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.

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.

To determine the behavior of transthyretin (TTR) in blood circulation, TTR purified from normal subjects’ plasma was injected to rats, and blood and urine were collected time dependently. Although TTR in plasma was proven to be a predominantly cysteine (Cys) conjugated form by electrospray ionization mass spectrometry (ESI-MS) analysis, it was gradually converted into free, 32 Da (dihydroxylation), 80 Da (phosphorylation), and 306 Da (glutathionylation), increased forms in molecular weight of TTR. The plasma levels of TTR were decreased in a time-dependent manner with the half life of 72.4 min. No secretion of TTR into the urine was observed by ESI-MS. In conclusion, this method can be simply performed without loading a radioactive molecule to the targeted protein. It offers a possibility to determine natural protein behaviors in the blood stream.

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.