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Supramolecular one-step self-assembly of dimanganese decacarbonyl, diaryl diselenide, and linear dipyridyl ligands (L = pyrazine (pz), 4,4'-bipyridine (bpy), and trans -1,2-bis(4-pyridyl)ethylene (bpe)) has resulted in the formation of selenolato-bridged manganese(I)-based metallorectangles. The synthesis of tetranuclear Mn(I)-based metallorectangles [{(CO) 3 Mn(μ-SeR) 2 Mn(CO) 3 } 2 (μ-L) 2 ] ( 1 - 6 ) was facilitated by the oxidative addition of diaryl diselenide to dimanganese decacarbonyl with the simultaneous coordination of linear bidentate pyridyl linker in an orthogonal fashion. Formation of metallorectangles 1 - 6 was ascertained using IR, UV-vis, NMR spectroscopic techniques, and elemental analyses. The molecular mass of compounds 2 , 4 , and 6 were determined by ESI-mass spectrometry. Solid-state structural elucidation of 2 , 3 , and 6 by single-crystal X-ray diffraction methods revealed a rectangular framework wherein selenolato-bridges and pyridyl ligands define the shorter and longer edges, respectively. Also, the guest binding capability of metallorectangles 3 and 5 with different aromatic guests was studied using UV-vis absorption and emission spectrophotometric titration methods that affirmed strong host-guest binding interactions. The formation of the host-guest complex between metallorectangle 3 and pyrene has been explicitly corroborated by the single-crystal X-ray structure of 3 •pyrene. Moreover, select metallorectangles 1 - 4 and 6 were studied to explore their anticancer activity, while CO-releasing ability of metallorectangle 2 was further appraised using equine heart myoglobin assay.

To reveal the protective effect of Terminalia chebula Retz (TCR) on cardiotoxicity induced by radix of Aconitum kusnezoffii Reichb (AKR). Control, AKR, AKR-TCR 1:3, AKR-TCR 1:1, AKR-TCR 3:1 and TCR-prepared AKR groups were set up. After treatment, the heart tissues were observed by H&E staining and transmission electron microscope. Serum myoglobin (MB) and troponin (cTn) were detected by ELISA. UPLC-Q Exactive/MS analysis was performed to detect the metabolic difference among the groups. ELISA results showed that the MB and cTn values of AKR group were significantly higher than Control group (P<0.05), while those of the other groups were lower than AKR group. TCR-prepared AKR group had similar MB and cTn contents to the Control group. Histopathological examination also indicated better detoxifying effects in the TCR-prepared AKR and AKR-TCR 1:1 group. The serum metabolomics analysis showed obvious distinction between the AKR and Control groups, while AKR-TCR combination reversed the metabolomics changes induced by AKR. Through multivariate statistical analysis, 9 metabolic markers related to energy, nucleic acid and amino acid metabolism were identified. Conclusively, AKR-induced cardiotoxicity may be related to energy, nucleic acid and amino acid metabolism, and TCR can reduce the cardiotoxicity by regulating the relative metabolism pathways.

Background: Following public concern on the use of synthetic food antioxidants, there is an increasing demand for the application of mixed or purified natural antioxidants to maintain quality of meat products quality during storage. The aim of this research was to investigate the effect of ethanolic extract of hawthorn berry, compared to butylated hydroxylanisole (BHA), on lipid peroxidation, myoglobin oxidation, protein electrophoresis pattern, consistency and firmness of minced pork during refrigeration at 4 °C, and to identify the relationship between chemical modifications and consistency variation., Results: After 6 days of refrigeration it was found that the thiobarbituric acid reactive substances value of minced pork containing 200 mg GAE kg -1 total phenolics in minced meat (200 HP) was significantly lower (0.1543 ± 0.006 mg) compared to BHA-treated meat. The ratio of oxymyoglobin to metmyoglobin in treated minced pork was respectively 0.845 for 200 HP and 0.473 for BHA-treated minced meat. Concentrations of 100 HP or 300 HP will generate statistically higher firmness than BHA in minced pork., Conclusion: Hawthorn berry ethanolic extract was more effective than BHA in reducing lipid oxidation and protein degradation, for maintaining firmness and consistency of minced pork during 6 days of refrigeration at 4 °C. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Iron is an important trace element for proper cell functioning. It is present in cytochromes, hemoglobin and myoglobin (Mb), where it binds to oxygen. It is also an electron acceptor in the respiratory chain. Mb is an 18 kDa heme-protein, highly expressed in skeletal muscle and heart. The expression of several genes involved in the metabolism of iron is post-transcriptionally regulated by this element. Iron was shown to interfere with the polyadenylation step, modifying their poly (A) tail length and, as a consequence, their stability and translation rate. The aim of this study was to investigate whether iron supplementation or long and short-term restriction affects Mb gene and protein expression, as well as Mb mRNA poly(A) tail length, in cardiac and skeletal muscles of rats. Long-term iron restriction caused an increase in Mb gene and protein expression in Soleus muscle. No changes were observed in extensor digitorum longus muscle and heart. Short-term iron supplementation after iron deprivation did not alter Mb gene expression and mRNA poly(A) tail length in all tissues studied. These results indicate that Mb gene and protein expression is upregulated in response to iron deprivation, an effect that is tissue-specific and seems to occur at transcriptional level.

Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80(low)CD11b(high)Ly6b(high)CD206(low) kidney macrophages were dominant, whereas by day 8, F4/80(high)CD11b(+)Ly6b(low)CD206(high) cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80(low)CD11b(high)Ly6b(high)CD206(low) macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease., (Copyright © 2015 by the American Society of Nephrology.)

Xanthine oxidase (XO), a free radical-generating enzyme, is involved in tissue damage produced during exhaustive exercise. Our aim was to test whether allopurinol, a powerful inhibitor of XO, may be effective in preventing exercise-induced tissue damage in soccer players. Twelve soccer players were randomized into two experimental groups. One received allopurinol, before a match of the premier Spanish Football League, and the other placebo. Allopurinol prevented the exercise-induced increase in all the markers of skeletal muscle damage analyzed: creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and myoglobin. Creatine kinase-MB isoenzyme and highly sensitive troponin T, specific biomarkers of myocardial injury, increased significantly in the placebo but not in the allopurinol-treated group after the football match. We also found that the exercise-induced lipid peroxidation, as reflected by malondialdehyde measurements, was prevented after allopurinol administration. However, inhibition of XO did not prevent the increment in the activity of alanine aminotransferase found after the match. No changes in the serum gamma glutamyltransferase activity was found after the match on either the placebo and the allopurinol groups. These two enzymes were determined as biomarkers of liver injury. Allopurinol represents an effective and inexpensive pharmacological agent to prevent tissue damage in soccer players., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The mechanisms by which general anesthetics, including xenon and nitrous oxide, act are only beginning to be discovered. However, structural approaches revealed weak but specific protein-gas interactions., Methods: To improve knowledge, we performed x-ray crystallography studies under xenon and nitrous oxide pressure in a series of 10 binding sites within four proteins., Results: Whatever the pressure, we show (1) hydrophobicity of the gas binding sites has a screening effect on xenon and nitrous oxide binding, with a threshold value of 83% beyond which and below which xenon and nitrous oxide, respectively, binds to their sites preferentially compared to each other; (2) xenon and nitrous oxide occupancies are significantly correlated respectively to the product and the ratio of hydrophobicity by volume, indicating that hydrophobicity and volume are binding parameters that complement and oppose each other's effects; and (3) the ratio of occupancy of xenon to nitrous oxide is significantly correlated to hydrophobicity of their binding sites., Conclusions: These data demonstrate that xenon and nitrous oxide obey different binding mechanisms, a finding that argues against all unitary hypotheses of narcosis and anesthesia, and indicate that the Meyer-Overton rule of a high correlation between anesthetic potency and solubility in lipids of general anesthetics is often overinterpreted. This study provides evidence that the mechanisms of gas binding to proteins and therefore of general anesthesia should be considered as the result of a fully reversible interaction between a drug ligand and a receptor as this occurs in classical pharmacology.

The effect of a whey protein- and carbohydrate (CHO)-enriched diet on the rate of muscle glycogen resynthesis after a soccer match was examined. Sixteen elite soccer players were randomly assigned to a group ingesting a diet rich in carbohydrates and whey protein [CHO, protein, and fat content was 71, 21, and 8E%, respectively; high content of carbohydrates and whey protein (HCP), n = 9] or a group ingesting a normal diet (55, 18, and 26E%; control [CON], n = 7) during a 48-h recovery period after a soccer match. CON and three additional players carried out a 90- and 60-min simulated match without body contacts (SIM90 and SIM60). Muscle glycogen was lowered (P < 0.05) by 54, 48, 53, and 38% after the matches in CON, HCP, SIM90, and SIM60, respectively. Glycogen resynthesis during the first 48 h after the match was not different between CON and HCP, whereas glycogen resynthesis was slower (P < 0.05) during the first 24 h after SIM60 than SIM90 (2.88 ± 0.84 vs 4.32 ± 0.54 mmol/kg dw/h). In HCP, glycogen content in type II muscle fibers was still lowered 48 h after the match. In conclusion, glycogen resynthesis 48 h after a soccer match is not elevated by ingestion of a HCP diet. Furthermore, glycogen resynthesis does not appear to be impaired by body contacts during a match., (© 2011 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Unlabelled: Our overall objective was to better understand the effects of added pyruvate on enhanced beef color stability. The 2 possible mechanisms assessed were the role of pyruvate in lipid oxidation and direct interaction between pyruvate and beef myoglobin. Microsomes were incubated with pyruvate at pH 5.6, 25 °C, and lipid oxidation was measured hourly for 3 h. Bovine oxymyoglobin at pH 5.6 was incubated with pyruvate and used to quantify both redox stability (metmyoglobin formation) and pyruvate-myoglobin adduction using mass spectrometry analysis. Surface color and lipid oxidation were measured on ground beef patties stored for 6 d in polyvinyl chloride over-wrap (PVC) or high oxygen. Addition of pyruvate to microsomes decreased lipid oxidation compared with controls (P < 0.05). Conversely, no effect on myoglobin was observed (no changes in redox stability and no peaks corresponding to pyruvate were observed; P > 0.05). However, pyruvate increased color stability and decreased lipid oxidation of ground beef patties packaged in PVC and high oxygen. Pyruvate decreased nitric oxide metmyoglobin-reducing capacity and oxygen consumption of patties compared with controls (P < 0.05). This research suggests that pyruvate may improve beef color stability primarily through its antioxidant effect on lipids., Practical Application: Discoloration of meat often results in significant revenue loss. This study suggests that pyruvate can improve the color stability of patties packaged in high oxygen and PVC primarily through its antioxidant effect on lipids., (© 2012 Institute of Food Technologists®)

Dimethyl sulfoxide (DMSO) is an organic compound widely used as solvent in biological studies and as vehicle for drug administration. DMSO has been shown to possess several biological effects, including antioxidant, anti-inflammatory, antinociceptive effects, and it has been proposed to be therapeutic in several disorders, such as gastrointestinal diseases, rheumatologic diseases, and for the treatment of several manifestations of amyloidosis. To better define the biological profile of DMSO, we investigated its effect on an in vivo model of acute myocardial infarction in rats, caused by left anterior descending coronary artery ligation. Our results show that pretreatment of rats with intraperitoneal (ip) DMSO (500 microL/Kg) for 3 consecutive days before left anterior descending coronary artery ligation significantly (P < 0.05) reduced cardiac damage from 18.75 +/- 4.88% (n = 12) to 4.46 +/- 2.01% (n = 8); serum levels of troponin I from 29.35 +/- 12.32 ng/mL (n = 8) to 2.95 +/- 1.32 ng/mL (n = 4); and serum levels of myoglobin from 46.86 +/- 10.35 ng/mL (n = 7) to 13.75 +/- 0.85 ng/mL (n = 4). Our data demonstrate that DMSO has a protective effect in a model of acute myocardial infarction in rats.

This study compared myoglobin (Mb) oxidation in lipid-free model systems containing iron and Type I (radical quenching) or Type II (metal chelating) antioxidants. Oxidation was measured as loss of oxymyoglobin (MbO(2)) during 0 to 24 h holding at 22 degrees C. Sodium tripolyphosphate (STPP) demonstrated iron-binding ability at all concentrations tested (88% and 21% added iron bound at 1 and 0.05 mg/mL, respectively). Iron chelation was observed for phytic acid only at the highest concentration (9.5% bound at 1 mg/mL phytate). Neither Type I antioxidant (rosmarinate or eugenol) demonstrated any iron chelating ability (<0.5% bound). In presence of iron, Type I antioxidants had a significant (P < 0.05) prooxidant effect (54.7% retention of MbO(2) in control, 9.5% and 37.5% retention in rosmarinate and eugenol samples, respectively). The Type II antioxidants (STPP and phytate) were more effective inhibitors (P < 0.05) of Mb oxidation than Type I antioxidants, (68.7% and 61.1% for STPP and phytate, respectively). Type I antioxidants were capable of rapid reduction of ferric iron to the ferrous form, as measured by the ferrozine assay. This strong reducing ability accounted for the prooxidant effects of rosmarinic acid and eugenol, since ferrous iron is the form associated with generation of oxygen radicals, and subsequent Mb oxidation. Type II antioxidants chelated and thus prevented the oxidizing effect of added ferrous iron. Mb oxidation can proceed rapidly (within 15 min) in the presence of iron and the absence of lipid, especially if reducing compounds such as rosmarinic acid or eugenol are also present to maintain iron in an active ferrous form.

To find out the changes in the internal dynamics and function of proteins as a consequence of their binding interactions with guanidine hydrochloride (GdnHCl), laser flash photolysis and optical absorption methods have been used to study the dynamic events in the horse myoglobin-CO complex (MbCO) in the presence of subdenaturing concentrations of GdnHCl at pH 7, 22 degrees C. The rate coefficients for geminate rebinding and bimolecular rebinding (k(on)) were measured by laser photolysis of CO in MbCO, and the CO dissociation rate (k(off)) was determined by the CO replacement method using hexacyanoferrate ion or NO. Starting from the native-state condition, the values of k(on) and k(off) decrease by approximately 1.4 (+/-0.1)-fold in the presence of 0.1-0.3 M GdnHCl, and then increase at higher concentrations of the denaturant. This has been taken as evidence for internal motional constraints and increased stability of the protein in the subdenaturing region giving rise to gated entry of the photolyzed CO from the solvent. The rate for geminate rebinding does not show any decrease in the rate versus GdnHCl concentration plots. The values for the activation enthalpy for the CO dissociation reaction and the entropy loss relative to the native-state entropy, both measured as a function of GdnHCl concentration, indicate that the protein is indeed stabilized under subdenaturing conditions. Analyses of thermal unfolding transitions of myoglobin in the presence of different concentrations of GdnHCl indicate that the stability of this protein extracted from the linear free energy model is approximately 3-4 kcal mol(-1) less than the true stability. The results indicate the appropriateness of the denaturant binding model for the analysis of GdnHCl-induced unfolding data, and provide a value of 7.9 kcal mol(-1) as the true stability of the protein.

Nonenzymatic glycation of proteins is associated with the long-term diabetes complication. The aim of this work was to examine in vitro the infuence of metformin on glycated proteins formation by mass spectrometry (ESI/MS, LC/MS/MS) and spectrofluorimetric method. Obtained results suggest that metformin dose-dependently inhibits early stage of Maillard reaction, although with the weaker potency than known glycation inhibitor aminoguanidine.

We investigated whether the lower fatigue resistance in smokers than in nonsmokers is caused by a compromised muscle oxidative metabolism. Using calibrated histochemistry, we found no differences in succinate dehydrogenase (SDH) activity, myoglobin concentration, or capillarization in sections of the vastus lateralis muscle between smokers and nonsmokers. The relationship between fatigue resistance and SDH activity in nonsmokers (r = 0.93; p = 0.02) is absent in smokers. This indicates that the lower muscle fatigue resistance of smokers can likely be attributed to causes other than differences in oxidative metabolism and capillarization.

Trehalose, a disaccharide present in many nonmammalian species, protects cells against various environmental stresses. Trehalose has recently been shown to decrease aggregate formation and toxicity in cell models and to alleviate amyloid-induced diseases. The aim of our study was to use two amyloid-forming proteins, i.e., W7FW14F apomyoglobin and insulin, as model systems to elucidate the molecular mechanism by which trehalose affects the amyloid aggregation process and to investigate further its therapeutic potential. Protein aggregation was examined by far-UV circular dichroism, UV absorption, thioflavin T fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, atomic force microscopy, and Fourier transform infrared spectroscopy. Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. We found that trehalose does not inhibit protein aggregation but acts at different stages of the fibrillization process depending on the protein model used. In fact, trehalose dose-dependently inhibited fibril formation in the W7FW14F apomyoglobin model and increased the lag phase in the insulin model. In both cases, trehalose caused accumulation of toxic oligomeric species. The results suggest that trehalose may favor or inhibit the formation of "on-pathway" or "off-pathway" oligomeric intermediates depending on the nature of the aggregating protein.

Objective: Accumulating evidence suggests that a hypercoagulable state influences early graft failure after off-pump coronary artery bypass (OPCAB). We hypothesized that regional myocardial ischemia caused by obligatory periods of coronary occlusion during OPCAB is an important trigger for this prothrombotic state., Methods: Using a series of biomarkers, 60 consecutive patients undergoing OPCAB were monitored for myocardial injury (myoglobin), inflammation (TNF-alpha, IL-8) and thrombosis (thrombin generation-F1.2, contact activation pathway-FXII-a, platelet derived microparticles-via flow cytometry). The transcardiac gradients of these markers were determined by assaying both arterial and coronary sinus blood just after protamine administration. Intramyocardial pH was monitored continuously during coronary occlusion in a subset (N=30 grafts, 11 patients). The influence of management strategies affecting hemostasis (e.g. antiplatelet therapy, anti-fibrinolytics, peak activated clotting time (ACT) during heparinization) was analyzed., Results: Ischemic injury, depicted by the transcardiac myoglobin gradient, significantly correlated with intramyocardial acidosis during coronary occlusion (R=0.96, p<0.0001) and predicted the transcardiac gradients of TNF-alpha (R=0.83, p<0.001) and F1.2 (R=0.72, p<0.0001). Transcardiac F1.2 strongly correlated with TNF-alpha (R=0.73, p=0.01) and IL-8 (R=0.51, p=0.02). Patients receiving aprotinin (N=20) showed significantly lower transcardiac gradients for myoglobin (4.1+/-7.5% vs 72.9+/-108.8% change, p=0.002), F1.2 (31+/-37% vs 89+/-149%, p=0.03), FXII-a (2.6+/-4.1% vs 19.2+/-34%, p=0.04) and microparticles (7+/-3.9% vs 12.9+/-8%, p=0.01)., Conclusions: Strong correlations between myocardial ischemia and the transcardiac gradients of markers for inflammation and thrombosis suggest that even brief episodes of coronary occlusion in the beating heart may have pathophysiologic consequences. Aprotinin, but not other factors that influence the coagulation system, appears to mitigate this process during OPCAB.

The long-term efficacy and safety of HMG-CoA reductase inhibitors (statins) have been established in large multicenter trials. Inhibition of this enzyme, however, results in decreased synthesis of cholesterol and other products downstream of mevalonate, such as CoQ10 or dolichol. This was a randomized double-blind, placebo-controlled study that examined the effects of CoQ10 and placebo in hypercholesterolemic patients treated by atorvastatin. Eligible patients were given 10mg/day of atorvastatin for 16 weeks. Half of the patients (n=24) were supplemented with 100mg/day of CoQ10, while the other half (n=25) were given the placebo. Serum LDL-C levels in the CoQ10 group decreased by 43%, while in the placebo group by 49%. The HDL-C increment was more striking in the CoQ10 group than in the placebo group. All patients showed definite reductions of plasma CoQ10 levels in the placebo group, by 42%. All patients supplemented with CoQ10 showed striking increases in plasma CoQ10 by 127%. In conclusion atorvastatin definitely decreased plasma CoQ10 levels and supplementation with CoQ10 increased their levels. These changes in plasma CoQ10 levels showed no relation to the changes in serum AST, ALT and CK levels. Further studies are needed, however, for the evaluation of CoQ10 supplementation in statin therapy.

Myoglobin (Mb) redox stability affects meat color and is compromised by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Pork lipids are generally more unsaturated and would be expected to oxidize readily and produce more oxidation products than beef. Supranutritional supplementation of vitamin E improves Mb redox stability of beef but not pork. The present study investigated HNE-induced redox instability in porcine and bovine myoglobins at 4 degrees C and pH 5.6. Oxymyoglobin (OxyMb) was incubated with HNE (0.075 mM porcine OxyMb + 0.5 mM HNE; 0.15 mM bovine OxyMb + 1.0 mM HNE). In porcine Mb, only monoadducts formed via Michael addition were detected after 72 h, whereas in bovine Mb both mono- and diadducts were identified. LC-MS-MS identified four histidine residues (His 36, 81, 88, and 152) of bovine Mb that were readily adducted by HNE, whereas in porcine Mb only two histidine residues (His 24 and 36) were adducted. These results suggested that the primary structure of bovine Mb predisposes it to greater nucleophilic attack by HNE and subsequent adduction than is suffered by porcine Mb.

Background: Insulin-like growth factor 1 (IGF1) is an important determinant of muscle mass because it promotes growth and suppresses protein degradation. IGF1 is decreased in rheumatoid arthritis and juvenile idiopathic arthritis because its synthesis is inhibited by inflammation. In parallel, glucocorticoids induce IGF1 resistance and add to muscle degradation., Objective: To investigate the influence of anti-tumour necrosis factor antibody treatment (anti-TNF) with adalimumab on levels of myoglobin (degradation marker) and IGF1 in patients with rheumatoid arthritis with and without prednisolone treatment., Methods: Subcutaneous adalimumab was given to 32 patients with longstanding rheumatoid arthritis (16 with and 16 without prednisolone) in a longitudinal study. IGF1, IGF1 binding protein 1 (IGFBP-1), IGFBP-3, and myoglobin were measured by enzyme linked immunosorbent assay., Results: Rheumatoid patients had normal serum myoglobin. Patients on prednisolone had higher myoglobin than patients not receiving prednisolone, indicating increased muscle degradation. On treatment with anti-TNF, myoglobin levels did not change in either patient group. Serum IGF1 was increased in patients with v without prednisolone, indicating IGF1 resistance (mean (SEM): 221 (23) v 122 (14) microg/l, p<0.001). Adalimumab treatment decreased the raised IGF1 levels in patients with prednisolone, so that after 12 weeks of treatment they reached the level of patients without prednisolone. Serum IGFBP-1 and IGFBP-3 did not differ in the two groups, and anti-TNF did not change these concentrations., Conclusions: Anti-TNF antibody treatment over 12 weeks improved glucocorticoid induced IGF1 resistance without influencing myoglobin and IGF1 binding proteins. Thus, in rheumatoid patients on glucocorticoids with generally decreased muscle mass anti-TNF treatment with adalimumab has favourable effects.

To expand the repertoire of artificial enzymes that are constructed by replacing the natural prosthetic group of hemoproteins with non-natural cofactors, we examined incorporation of a non-porphyrinic ligand (1) into the heme-pocket of apomyoglobin in a non-covalent fashion. Ligand 1 is a highly conjugated 1,10-phenanthroline derivative, which shares some structural features with protoporphyrin IX; for example, molecular size and arrangement of hydrophobic and anionic parts. Addition of apomyoglobin to a solution of 1 induces clear changes in the absorption spectrum of 1, suggesting one-to-one incorporation of 1 into the heme cavity of apomyoglobin with an affinity of 6.3 x 10(6)M(-1). We found that the hydrolytic activity of apomyoglobin toward p-nitrophenyl hexanoate was greatly suppressed because of the incorporation of 1 into the heme-pocket.

Nitric oxide (NO) signal transduction occurs through guanylyl cyclase-coupled receptors, which exist in both cytosolic and membranous locations. It has recently been reported from experiments using heart tissue that the membrane-associated receptor has enhanced sensitivity to NO. Owing to its potential importance, we tested this finding using a method of applying NO in known, constant concentrations. The results showed that the concentration-response curves for receptor activation in cytosolic and membrane preparations of two different tissues (cerebellum and platelets) were indistinguishable. In all cases, half-maximal activation required about 1 nM NO and the curves had Hill coefficients of close to 1. The differential sensitivity reported for the heart is attributed to NO being scavenged by myoglobin in the cytosol, but not in the membrane fraction.

Metmyoglobin (Mb) was glycated by glucose in a non-enzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO2 and GMbO2, respectively. H2O2-induced iron release was significantly higher from GMbO2 than that from MbO2. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H2O2 was studied. GMbO2 degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO2. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H2O2 with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO2. Compared to MbO2, GMbO2 was rapidly autooxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO2. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H2O2. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

Background: Coronary patency is important for short- and long-term outcome after myocardial infarction. Serum myoglobin concentration is a sensitive marker of myocardial damage and its specificity can be improved by simultaneous measurement of carbonic anhydrase III, a skeletal muscle marker. In the present study we evaluated the role of myoglobin/carbonic anhydrase III ratio as a non-invasive marker of reperfusion., Methods: We measured myoglobin, carbonic anhydrase III and creatine kinase MB-fraction release serially in 29 patients with acute myocardial infarction treated with thrombolysis and in 28 patients treated with primary coronary angioplasty., Results: Thrombolytic therapy was followed by a 9.1+/-2.2-fold increase in myoglobin and 10.8+/-3.3-fold increase in creatine kinase MB-fraction during the first hour of treatment, while carbonic anhydrase III remained unchanged. The peak value of myoglobin/carbonic anhydrase III ratio was found at 2 h and that of creatine kinase MB-fraction at 8 h after thrombolysis. Knowledge of the reperfusion time point during primary angioplasty and follow-up of cardiac markers revealed that cut-off points of 3 and 10 h for the peak values of myoglobin/carbonic anhydrase III ratio and creatine kinase MB-fraction can be used as indicators for reperfusion, respectively. Myoglobin/carbonic anhydrase III ratio measured before treatment and at 2 and 4 h after the onset of treatment screened 23 of those 25 patients with probable reperfusion after thrombolysis., Conclusions: We conclude that measuring myoglobin/carbonic anhydrase III ratio during the first hours after initiation of thrombolysis may be useful in evaluating the success of reperfusion after acute myocardial infarction.

A protein-cleaving catalyst highly selective for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin (Mb) was designed by attaching the Cu(II) or Co(III) complex of cyclen to a binding site searched by a combinatorial method using peptide nucleic acid monomers as building units. Various linkers were inserted between the catalytic Co(III) center and the binding site of the Mb-cleaving catalyst. Kinetic data revealed catalytic turnover of the Mb cleavage by the Cu(II) or Co(III) complex. MALDI-TOF MS revealed cleavage of the polypeptide backbone of Mb at selected positions. N-Terminal sequencing of the cleavage products identified the cleavage site and provided evidence for the hydrolytic nature of the Mb cleavage. Various chelating ligands were tested as the ligand for the Co(III) center of the Mb-cleaving catalyst. Among the nine chelating ligands examined, only cyclen and its triaza-monooxo analogue manifested catalytic activity.

Background: Vitamin E has a strong antioxidant capacity, and has been used in several ischemia-reperfusion studies. The aim of this study was to investigate the effects of water-soluble vitamin E (alpha-tocopherol) on myocardial protection during coronary artery surgery., Methods: Water soluble vitamin E (100 mg) in tepid saline (n = 14) or tepid saline alone (n = 16) was administered into the coronary arteries at the end of aortic cross-clamping. Cardiac troponin-I (cTn-I), MB-isoenzyme of creatine kinase (CK-MB), myoglobin, blood gas, and lactate levels in systemic and coronary sinus blood and hemodynamic variables were assessed during and after the operation., Results: Eight hours after reperfusion, cTn-I levels increased to 3.06 +/- 1.8 ng/ml and 6.97 +/- 3.9 ng/ml in the vitamin E group and control group, respectively (p = 0.01). Coronary sinus lactate concentration was 2.68 +/- 0.5 mmol/l in the vitamin E group and 4.01 +/- 1.5 mmol/l in the control group 60 minutes after reperfusion (p = 0.027)., Conclusions: Administration of vitamin E into the coronary arteries before removal of the aortic cross-clamp can reduce myocardial cell injury and protect the myocardium from ischemia-reperfusion injury.

The effects of aqueous ethanol or 2,2,2-trifluoroethanol media on the structure of sperm whale myoglobin have been investigated by absorption, CD, and NMR spectra. The structural properties of myoglobin such as heme environments, helix contents, protein folding, and interactions between heme and the protein moiety have been sharply manifested in these spectra. The characterization demonstrated that alcohol-induced conformational change of myoglobin depends on the nature of alcohol and its concentration. It was shown for the first time that, upon the alcohol-induced denaturation of myoglobin, heme is released from partially denatured protein of which helix contents is altered by only about 20% relative to that of native state. Myoglobin has shown to unfold and refold reversibly by controlling the alcohol concentration. Novel methods for the preparation of apomyoglobin and in situ reconstitution of apomyoglobin with heme, based on the alcohol-induced denaturation of the protein, were presented.

We report here that a native folded holo-myoglobin, when incubated with a synthetic amyloidogenic peptide in aqueous solutions, forms fibrils. These fibrils took a cross-beta form (inter-strand spacing: 4.65 A and inter-sheet spacing: 10.65 A) and bound the amyloidophilic dye Congo red as did the authentic amyloid fibrils. In contrast such fibril formation of myoglobin did not occur in the absence of the peptide. These results suggest the possibility that inter-molecular interaction of native protein with the amyloidogenic peptide trigger the amyloid formation even for the non-pathogenic native protein like myoglobin, which itself exists as a globular form, under certain conditions.

The red color of muscle is principally due to the presence of oxymyoglobin. Oxidation of heme iron from the ferrous to the ferric state produces a brownish color, which consumers find undesirable. The aim of this study was to use enzymic and nonenzymic antioxidants to simulate in situ muscle antioxidation reactions in order to understand better the mechanism by which the iron redox cycle catalyzes membrane lipid peroxidation and oxymyoglobin oxidation. The inclusion of superoxide dismutase (SOD) in the model system decreased oxymyoglobin oxidation by 10% without affecting lipid peroxidation. Addition of catalase decreased oxymyoglobin oxidation by approximately 40% but not lipid peroxidation. Increasing the ceruloplasmin concentration inhibited lipid peroxidation but increased oxymyoglobin oxidation, which was inhibited by SOD and catalase. Conalbumin (50 microM), a specific iron chelator, inhibited peroxidation and oxymyoglobin oxidation by almost 50%. The addition of the antioxidant catechin (500 microM) decreased lipid peroxidation by 90% but oxymyoglobin oxidation by only 50%. Feeding turkeys with vitamin E at several levels significantly increased the alpha-tocopherol level of membranes, thus preventing oxymyoglobin and lipid oxidation. In conclusion, oxymyoglobin stability in the model system was affected by two pathways: (a) oxygen active species, such as O(2)*(-), H(2)O(2), HO*, and ferryl, generated during autoxidation of myoglobin and oxidation of ferrous ions and ascorbic acid; and (b) lipid radicals, such as ROO*, RO*, and hydroperoxides, generated during lipid peroxidation. Maximum inhibition could be achieved only by introducing inhibitors of both pathways into the system.

The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.

The formation of oxidized low density lipoprotein (LDL) is believed to play a significant role in the pathogenesis of atherosclerosis. Myoglobin in the presence of H(2)O(2) has been shown to catalyze LDL oxidation in vitro. It is established that an oxidatively altered form of myoglobin (Mb-H), which contains a prosthetic heme covalently crosslinked to the apoprotein, is a major product in the reaction of native myoglobin with peroxides. In the current study, we have shown for the first time that Mb-H, in the absence of exogenously added peroxides, oxidizes LDL and purified lipids, as determined by the formation of conjugated dienes, lipid peroxides, and thiobarbituric acid reactive substances. Moreover, the rate of oxidation of pure phosphatidylcholine by Mb-H was found to be at least sevenfold greater than that observed for native myoglobin. The current study strongly suggests a role for Mb-H in the lipid peroxidation observed with myoglobin., (Copyright 2000 Academic Press.)

This study investigates the potential basis for enhancement of oxymyoglobin (OxyMb) oxidation by lipid oxidation products. Aldehydes known to be formed as secondary lipid oxidation products were combined with OxyMb in aqueous solution at 37 degrees C and pH 7.4. Metmyoglobin (MetMb) formation was greater in the presence of alpha,beta-unsaturated aldehydes than their saturated counterparts of equivalent carbon chain length. Additionally, increasing chain length from hexenal through nonenal resulted in increased MetMb formation (P < 0.05). Electrospray ionization mass spectrometry (ESI-MS) revealed that OxyMb incubated with 4-hydroxynonenal (HNE) at pH 7.4 at 37 degrees C yielded myoglobin molecules adducted with one to three molecules of HNE from 0.5 to 2 h of incubation, respectively. A prooxidant effect of HNE was noted at pH 7.4 but was not apparent at pH 5.6 when compared to the control (P < 0.05). This appeared to be due to rapid OxyMb autoxidation at this pH compared to pH 7.4. ESI-MS demonstrated that adduction of HNE to OxyMb occurred at pH 5.6. This research demonstrates that alpha, beta-unsaturated aldehydes accelerate OxyMb oxidation and appear to do so via covalent attachment.

To determine whether specific or nonspecific interactions between inhaled anesthetics and proteins are more likely to underlie anesthetic actions, analysis of hydrogen/tritium exchange was used to measure effects on the stability of two model proteins that had been previously shown to bind anesthetics specifically (bovine serum albumin) or only nonspecifically (myoglobin). The data indicated that stabilization of albumin correlated with the potencies of a wide range of anesthetic compounds significantly better than did destabilization of myoglobin. In addition, sensitivity to nonanesthetics, isoflurane stereoselectivity, and temperature and pressure effects all influenced the stabilization of bovine serum albumin, but not the destabilization of myoglobin, in a manner strongly supporting the premise that specific binding interactions with protein targets underlie anesthetic action. These observations significantly increase the likelihood that such interactions can be found and optimized.

We have demonstrated that N-terminal sequencing can be performed successfully despite boiling protein samples in the presence of urea under precise conditions, before loading them onto SDS-PAGE and transfer to polyvinylidene difluoride membrane. Using myoglobin as a test protein, we found that its ability to undergo N-terminal sequencing was not affected by the presence of urea provided "ultra-pure" urea was used. Consistent with this result, we verified that urea did not carbamylate myoglobin since its molecular mass was measured by mass spectrometry after electroelution of the protein band from the gel. These observations are useful for the study of integral membrane proteins, in particular to study their topology from proteolysis experiments, since heating in the presence of urea before SDS-PAGE reduces membrane protein aggregation [Soulié, S., Mo/ller, J.V., Falson, P., and le Maire, M. (1996) Anal. Biochem. 236, 363-364]. We show that the sequencing yield of a hydrophobic peptide from reticulum Ca2+-ATPase was more than doubled in the presence of urea in accord with the quantification of the Coomassie Blue staining of the gel and of the amount present on the polyvinylidene difluoride membrane. For three peptides of the gastric H+K+-ATPase, the sequencing yield after urea treatment increased almost threefold.

2,2,2-Trifluoroethanol (TFE)-induced nonnative alpha-helical structure in peptides and proteins has been extensively studied with circular dichroism (CD) spectroscopy. However, to date, complementary information from infrared (IR) spectroscopy has not been reported. Using both IR and CD spectroscopy, we demonstrate here that the TFE-induced nonnative alpha-helical structure in two beta-sheet-predominant proteins, beta-lactoglobulin and alpha-chymotrypsin, is unstable in comparison with those found in the alpha-helix-predominant proteins myoglobin and cytochrome c under identical conditions. IR spectra showed that, immediately after dissolution of the beta-sheet proteins in 50% (v/v) TFE, a strong amide I band component appears at 1654 cm-1 in H2O and at 1650 cm-1 in D2O, which is ascribed to alpha-helical structure. However, the intensities of the alpha-helical bands decrease as a function of time, concomitant with the appearance of two new band components near 1620 and 1695 cm-1 in H2O and 1612 and 1684 cm-1 in D2O, a typical IR spectral pattern for an intermolecular beta-sheet aggregate. Clear gels begin to develop within 30 min. No similar spectral changes were observed for the alpha-helical proteins. CD spectra suggested initially that the TFE-induced alpha-helix was retained in the gelled state. However, further analysis of the spectra, and Gaussian function modeling with basic spectra, indicated that the apparent alpha-helix signal was actually due to a combination of signals from intermolecular beta-sheet and residual alpha-helix. These results indicate that the TFE-induced nonnative alpha-helix structure in predominantly beta-sheet proteins is unstable and readily converts to an intermolecular beta-sheet aggregate., (Copyright 1998 Academic Press.)

To understand further the weak molecular interactions between inhaled anesthetics and proteins, we studied the character and dynamic consequences of halothane binding to bovine serum albumin (BSA) and myoglobin using photoaffinity labeling and hydrogen-tritium exchange (HX). We find that halothane binds saturably and with submillimolar affinity to BSA, but either nonspecifically or with considerably lower affinity to myoglobin. Titration of halothane binding with guanidine hydrochloride suggested more protection of binding sites from solvent in BSA as compared with myoglobin. Protection factors for slowly exchanging albumin hydrogens are increased in a concentration-dependent manner by up to 27-fold with 10 mM halothane, whereas more rapidly exchanging groups of albumin hydrogens have either unaltered or decreased protection factors. Protection factors for slowly exchanging hydrogens in myoglobin are decreased by halothane, suggesting destabilization through binding to an intermediate or completely unfolded conformer. These results demonstrate the conformation dependence of halothane binding and clear dynamic consequences that correlate with the character of binding in these model proteins. Preferential binding and stabilization of different conformational states may underlie anesthetic-induced protein dysfunction, as well as provide an explanation for heterogeneity of action.

2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native apomyoglobin.

Background: The cardiac isoform of troponin I (cTnI) is a myofibrillar protein highly specific for myocardial injury. We used a recently developed new-generation immunoassay with high analytical sensitivity to measure cTnI in patients diagnosed with hematologic malignancies, before chemotherapy and after an intermediate cumulative dose of anthracyclines. We hypothesized that measurement of cTnI with this sensitive method would provide evidence of myocardial injury in these patients., Methods: Sera from 115 individuals (60 healthy controls, 25 anthracycline-naive patients and 30 patients treated with intermediate cumulative doses of anthracyclines) were assessed for cTnI, creatine kinase MB (CKMB) mass and myoglobin. Radionuclide left ventricular ejection fraction (LVEF) was also determined., Results: Using this sensitive assay, detectable concentrations of cTnl were measured in the healthy population [mean, 19.5 pg/ml, 95% confidence interval (CI) 13.5-25.5 pg/ml]. Anthracycline-naive patients had cTnI mean values (36.5 pg/ml, 95% CI 25.1-47.9 pg/ml) that were significantly (P < 0.01) greater than those in the control group. cTnI was significantly (P < 0.00001) increased in anthracycline-treated patients (76.4 pg/ml, 95% CI 67.0-85.8 pg/ml) compared with both the anthracycline-naive patients and the controls. CKMB, myoglobin and LVEF were within the normal range in all patients., Conclusions: These data provide evidence for cardiac involvement in patients with hematological malignancies before and during the course of anthracycline chemotherapy. They suggest that detection of myocardial injury may be facilitated by measurement of cTnI with a highly sensitive assay.

Channichthyid icefishes of Antarctica are unique among adult vertebrates. All icefish species lack hemoglobin and red blood cells in their circulating blood. All icefishes examined to date also lack the intracellular oxygen-binding protein myoglobin (Mb) in their oxidative skeletal muscles. However, some icefish species do express Mb in their heart ventricles. It is unknown whether Mb in those species in which it is present represents an evolutionary relic or has functional significance. To address this problem, we compared mechanical performance of isolated, perfused hearts from two species of icefish in which Mb is either present (Chionodraco rastrospinosus) or is absent (Chaenocephalus aceratus). Hearts were challenged with increasing afterload (2.5-4.0 kPa) under conditions of defined basal flow (approximately 100 ml.min-1.kg-1), in both the presence and absence of 5 mM sodium nitrite, a Mb poison. Unlike hearts from C. aceratus, which were unable to maintain a constant cardiac output under pressure loading, those from C. rastrospinosus retained a constant flow up to 3.5 kPa afterload. At the upper range of power outputs, hearts of Mb-lacking C. aceratus display greater oxygen utilization than those of Mb-containing C. rastrospinosus. Poisoning of Mb significantly impaired the ability of C. rastrospinosus hearts to face pressure loading without reduction in flow, whereas those of C. aceratus were refractory to the treatment. The results strongly support a functional role for Mb in the former species.

Calcium repletion (2.0 mM) after 10 min of calcium depletion (pCa = 7) resulted in the loss of myoglobin, decreased contents of ATP and adeninucleotides. Adenosine in concentration 50-1000 mcM depressed the release of myoglobin and prevented depletion of ATP and adenine nucleotides during the "calcium paradox". Theophylline, a non-selective A1-receptors blocker did not alter either "calcium paradox" development or protective effect of adenosine. Dipiridamole, an inhibitor of transmembrane transport of adenosine, protected hearts against the "calcium paradox" depending on the concentration. Dipiridamole (50 mcM) abolished protective effect of high concentrations (100-1000 mcM) of adenosine. The results obtained prove that transmembrane transport of adenosine is more important than its interaction with receptors for its protective effect under the "calcium paradox".

The extent of oxygen release from two heme proteins, haemoglobin and myoglobin have been studied in the presence of trifluoperazine and chlorpromazine (5-1000 microM). At a molar ratio (drug:protein) of 1.5, the release of oxygen from haemoglobin was 4 and 15% in the presence of chlorpromazine and trifluoperazine respectively, while from myoglobin the corresponding values were 20 and 40%. The findings were attributed to the greater extent of local conformational change around tryptophan moieties of each of the proteins induced by trifluoperazine.

The effect of lactate on O2 binding properties of sperm whale and horse heart myoglobins (Mb) has been investigated at moderately acid pH (i.e. pH 6.5, a condition which may be achieved in vivo under a physical effort). Addition of lactate brings about a decrease of O2 affinity (i.e. an increase of P50) in sperm whale and horse heart myoglobins. Accordingly, lactate shows a different affinity for the deoxygenated and oxygenated form, behaving as a heterotropic modulator. The lactate effect on O2 affinity appears to differ for sperm whale and horse heart Mb, deltalogP50 being approximately 1.0 and approximately 0.4, respectively. From the kinetic viewpoint, the variation of O2 affinity for both myoglobins can be attributed mainly to a decrease of the kinetic association rate constant for ligand binding.

The contribution of specific packing to the stability of the sperm whale apomyoglobin intermediate has been studied by urea denaturation monitored by circular dichroism and fluorescence. Mutations disrupting native packing sites within the subdomain formed by the A, G and H helices destabilize the intermediate, in contrast to the conclusion drawn from earlier studies of pH-induced unfolding. Based on these results, the intermediate is proposed to be stabilized by both partially formed native-like tertiary, and non-specific hydrophobic interactions forming a subdomain folding intermediate. The results help to explain how the intermediate acquires its structure and stability.

To gain insight into the interactions between transcriptional factor proteins and DNA, the DNA-reactive drugs (+)-CC-1065 and pluramycin were used to target specific protein-DNA complexes. The structural features of the complex between the transcriptional activator Sp1 and the 21-base-pair repeat of the early promoter region of SV40 DNA were examined using hydroxyl-radical footprinting; (+)-CC-1065, a sequence-specific minor groove bending probe; and circularization experiments. The results show that the 21-base-pair repeat region has an intrinsically in-phase bent structure that is stabilized upon saturation Sp1 binding by protein-DNA and protein-protein interactions to produce a looping structure. The intercalating drug pluramycin was used to probe the structural details of the interaction between the TATA binding protein (TBP) and the TATA box DNA sequence. TBP, which directs initiation of RNA transcription, exhibits two-fold symmetry and apparently interacts with the TATA box in a symmetrical fashion. However, the interaction results in an asymmetric effect, in that transcription is initiated only in the downstream direction. Using pluramycin as a probe, it was determined that TBP binding to the human myoglobin TATA sequences enhances pluramycin reactivity at a site immediately downstream of the TATA box. The implications on transcriptional control of ternary complexes comprised of transcriptional factors, DNA, and DNA-reactive compounds will be presented.

The reaction of streptozotocin with oxymyoglobin was analyzed and compared with results using various compounds that spontaneously generate nitric oxide in solution.

The nature of the structural changes that apomyoglobin undergoes when subjected to hydrostatic pressure, ranging from atmospheric pressure to 2.4 kbar, has been investigated by steady-state fluorescence and frequency domain fluorometry. In particular, we have examined the intrinsic tryptophanyl emission and that of the extrinsic probe 1-anilino-8-naphthalenesulfonate (ANS) bound to apomyoglobin at neutral pH, as well as at strongly acidic high-salt conditions. Apomyoglobin at neutral pH undergoes a pressure-induced structural transition, which causes the disorganization of the heme binding region with a consequent ANS dissociation; a concomitant increase in solvent accessibility to the N-terminus of the macromolecule in which tryptophans are located is also observed. At 2.4 kbar, the tryptophanyl emission is not coincident with that of a fully solvent exposed residue, thus suggesting that the N-terminal region of the apomyoglobin molecule retains elements of organized structure. The spectroscopic properties of the structural state attained at 2.4 kbar and neutral pH are different from those of the acidic compact state. The acidic compact state of apomyoglobin undergoes a pressure-induced structural change that brings the tryptophanyl residues in contact with the solvent, but does not affect the ability to bind ANS.

The effect of tert-butyl hydroperoxide (tBH) on the nucleotide level in isolated cardiomyocytes from adult rats was examined. Incubation of myocytes with the hydroperoxide resulted in loss of both ATP and NAD from mitochondria and cytosol. In contrast, only the cytosolic level of NADP was increased. The effect on myocyte content of ADP and AMP was less pronounced. A significant increase in formation and release of adenosine also occurred in tBH-exposed cells. Treatment of cardiomyocytes with tBH resulted in a progressive decline of rod-shaped and viable cells, which was accompanied by an increase in numbers of hypercontracted and round myocytes in the incubation mixture. Electron microscopic examination showed an intense contraction of the cells. In summary our results demonstrate that plasma membrane damage and hypercontraction are early manifestations of tBH-induced injury to isolated cardiomyocytes. The depletion of ATP and formation of adenosine also suggest that degradation of adenine nucleotides may contribute to the cytotoxicity caused by tBH. Possible mechanisms involved in the ATP depletion are discussed.