Effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin.

In this work, we have studied the effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin (Mb), and by means of electrochemical techniques, we have shown that this reaction is undoubtedly affected following the interaction with the venom. Surface plasmon resonance, electrophoresis, UV-Vis, and circular dichroism showed that the interaction involves the attachment of some constituent of the venom to the protein, although not affecting its first and secondary structures. Mass spectra support this suggestion by showing the appearance of signals assigned to the Mb dimer and to a new species resulting from the interaction between Mb and the venom proteins. In addition, the mass spectra suggest the aromatic amino acids of myoglobin, mainly tryptophan and phenylalanine, are more exposed to the solvent medium upon the exposure to the venom solution. The results altogether indicate that the harmful effects of the venom of Crotalus basiliscus snake are likely connected to the blocking of the redox site of Mb.

Molecular Mechanisms and Novel Therapeutic Approaches to Rhabdomyolysis-Induced Acute Kidney Injury.

Rhabdomyolysis is a syndrome caused by injury to skeletal muscle that usually leads to acute kidney injury (AKI). Rhabdomyolysis has been linked to different conditions, including severe trauma and intense physical exercise. Myoglobin-induced renal toxicity plays a key role in rhabdomyolysis-associated kidney damage by increasing oxidative stress, inflammation, endothelial dysfunction, vasoconstriction, and apoptosis. New drugs that target the harmful effects of myoglobin have been recently developed, and some have been proven to be successful in animal models of acute renal failure secondary to rhabdomyolysis. This review aims to provide a comprehensive and updated overview of the pathological mechanisms of renal damage and describes new therapeutic approaches to this condition based on novel compounds that target key pathways involved in myoglobin-mediated kidney damage.

Enhancement of thermal stability and inhibition of protein aggregation by osmolytic effect of hydroxyproline.

A combination of spectroscopic, calorimetric, and microscopic studies to understand the effect of hydroxyproline on the thermal stability, conformation, biological activity, and aggregation of proteins has been investigated. Significantly increased protein stability and suppression of aggregation is achieved in the presence of hydroxyproline. For example, exceptional increase in the thermal stability of lysozyme up to 26.4 degrees C and myoglobin up to 31.8 degrees C is obtained in the presence of hydroxyproline. The increased thermal stability of the proteins is observed to be accompanied with significant rise of the catalytic activity. Hydroxyproline is observed to prevent lysozyme fibril formation in vitro. Fluorescence and circular dichroism studies indicate induction of tertiary structures of the studied proteins in the presence of hydroxyproline. Preferential hydration of the native state is found to be crucial for the mechanism of protein stabilization by hydroxyproline. We compared the effect of hydroxyproline to that of proline and observed similar increase in the activity and suppression of protein aggregation. The results demonstrate the use of hydroxyproline as a protein stabilizer and in the prevention of protein aggregation and fibril formation.

Role of myoglobin in regulating respiration.

The 1H NMR Val E11 signal provides a unique opportunity to observe carbon monoxide (CO) inhibition of Mb in the in vivo myocardium and to assess the functional role of Mb in regulating respiration. Upon carbon monoxide infusion, the MbO2 Val E11 signal at -2.76 ppm gradually disappears, and a new signal at -2.26 ppm, corresponding to MbCO, emerges. These signals yield the intracellular partial pressure of both O2 and CO and the extent of Mb inactivation, since CO binds more tightly to Mb than O2. Although contractile function decreases slightly to a steady state level, it shows no dose dependence on pCO. Up to 80% MbCO saturation, the contractile function remains at the steady state level. Neither the PCr concentration nor the oxygen consumption rate is significantly perturbed. Above 80% MbCO saturation, the oxygen consumption rate starts to decline. The experimental observations raise provocative questions about the functional role of Mb in the cell.

Pyruvate attenuates myoglobin in vitro toxicity.

Myoglobinuria is a complication of crush injury as well as substance abuse. This study examined whether pyruvate modified myoglobin in vitro renal toxicity. Renal slices from Fischer-344 rats were incubated for 120 min with 0-12 mg/ml myoglobin. In an initial study, gluconeogenesis was stimulated by the addition of 10 mM pyruvate during the final 30 min. In all other studies, renal slices were incubated with myoglobin in the presence of 0 or 10 mM pyruvate for 120 min. Myoglobin increased lactate dehydrogenase (LDH) release and this was not modified by the presence of pyruvate for the last 30 min of the incubation. Myoglobin toxicity was reduced by coincubation of myoglobin with pyruvate for 120 min. LDH leakage was increased 1.2-, 1.7-, and 1.8-fold above control by 4, 10, and 12 mg/ml myoglobin, compared to 1.2, 1.3, and 1.3 fold in slices coincubated with 10 mM pyruvate, respectively. Myoglobin diminished adenosine triphosphate (ATP) levels but pyruvate maintained a 5x higher level of ATP within the slices. Glucose (10 mM) provided protection only for the low concentration (4 mg/ml) of myoglobin. Myoglobin induced oxidative stress while pyruvate prevented the rise in lipid peroxidation and glutathione disulfides by myoglobin. Myoglobin diminished total glutathione levels in pyruvate-treated tissue, but glutathione levels remained higher than tissues incubated in the absence of pyruvate. These results indicate that pyruvate reduced toxicity by preventing oxidative stress and via a supply of an energy substrate.

Acute inhibition of myoglobin impairs contractility and energy state of iNOS-overexpressing hearts.

Elevated cardiac levels of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) have been implicated in the development of heart failure. The surprisingly benign phenotype of recently generated mice with cardiac-specific iNOS overexpression (TGiNOS) provided the rationale to investigate whether NO scavenging by oxymyoglobin (MbO2) yielding nitrate and metmyoglobin (metMb) is involved in preservation of myocardial function in TGiNOS mice. 1H nuclear magnetic resonance (NMR) spectroscopy was used to monitor changes of cardiac myoglobin (Mb) metabolism in isolated hearts of wild-type (WT) and TGiNOS mice. NO formation by iNOS resulted in a significant decrease of the MbO2 signal and a concomitantly emerging metMb signal in spectra of TGiNOS hearts only (DeltaMbO2: -46.3+/-38.4 micromol/kg, DeltametMb: +41.4+/-17.6 micromol/kg, n=6; P<0.05) leaving contractility and energetics unaffected. Inhibition of the Mb-mediated NO degradation by carbon monoxide (20%) led to a deterioration of myocardial contractility in TGiNOS hearts (left ventricular developed pressure: 78.2+/-8.2% versus 96.7+/-4.6% of baseline, n=6; P<0.005), which was associated with a profound pertubation of cardiac energy state as assessed by 31P NMR spectroscopy (eg, phosphocreatine: 13.3+/-1.3 mmol/L (TGiNOS) versus 15.9+/-0.7 mmol/L (WT), n=6; P<0.005). These alterations could be fully antagonized by the NOS inhibitor S-ethylisothiourea. Our findings demonstrate that myoglobin serves as an important cytoplasmic buffer of iNOS-derived NO, which determines the functional consequences of iNOS overexpression.

Nitric oxide prevents myoglobin/tert-butyl hydroperoxide-induced inhibition of Ca2+ transport in skeletal and cardiac sarcoplasmic reticulum.

Interaction of hydrogen peroxide or organic hydroperoxides with hemoproteins is known to produce oxoferryl hemoprotein species that act as very potent oxidants. Since skeletal and cardiac muscle cells contain high concentrations of myoglobin this reaction may be an important mechanism of initiation or enhancement of oxidative stress, which may impair their Ca2+ transport systems. Using skeletal and cardiac sarcoplasmic reticulum (SR) vesicles, we demonstrated by EPR the formation of alkoxyl radicals and protein-centered peroxyl radicals in the presence of myoglobin (Mb) and tert-butyl hydroperoxide (t-BuOOH). The low temperature EPR signal of the radicals was characterized by major feature at g = 2.016 and a shoulder at g = 2.036. In the presence of SR vesicles, the magnitude of the protein-centered peroxyl radical signal decreased, suggesting that the radicals were involved in oxidative modification of SR membranes. This was accompanied by SR membrane oxidative damage, as evidenced by accumulation of 2-thiobarbituric acid-reactive substances (TBARS) and the inhibition of Ca2+ transport. We have shown that nitric oxide (NO), reacting with redox-active heme iron, can prevent peroxyl radical formation activated by Mb/t-BuOOH. Incubation of SR membranes with an NO donor, PAPA/NO (a non-thiol compound that releases NO) at 200-500 microM completely prevented the t-BuOOH-dependent production of peroxyl radicals and formation of TBARS, and thus protected against oxidative inhibition of Ca2+ transport.

A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure.

Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.

19F nuclear magnetic resonance studies of free calcium in heart cells.

19F nuclear magnetic resonance is used in conjunction with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBapta), a fluorinated calcium chelator, to report steady-state intracellular free calcium levels ([Ca2+]i) in populations of resting, quiescent, isolated adult heart cells. 31P nuclear magnetic resonance shows that 5FBapta-loaded cells maintain normal intracellular high-energy phosphates, pH, and free Mg2+. The intracellular free calcium concentration of well perfused, isolated heart cells is 61 +/- 5 nM, measured with 5FBapta, which has a dissociation constant (Kd) for calcium chelation of 500 nM. A similar value is obtained with Quin-MF, another fluorinated calcium chelator with Kd and maximum calcium sensitivity at 80 nM. We find that the steady-state level of intracellular free calcium is increased by decreased extra-cellular sodium concentration, omission of extracellular magnesium, decreased extracellular pH, hyperglycemia, and upon treatment with lead acetate. Further, extracellular ATP caused a large transient increase in [Ca2+]i. Thus, while heart cells maintain a very low level of intracellular free Ca2+, acute alterations in extracellular environment can cause derangement of calcium homeostasis, resulting in measurable increases in [Ca2+]i.

NADH fluorescence of isolated ventricular myocytes: effects of pacing, myoglobin, and oxygen supply.

Endogenous fluorescence was used to measure the extent of reduction of mitochondrial NAD in individual, isolated rat cardiac myocytes. NAD reduction was determined from emitted fluorescence at 415 and 470 nm during brief epi-illumination at 365 nm. NAD reduction of resting myocytes, superfused with medium equilibrated with 95% O2/5% CO2, was 27 +/- 3% (SE) (n = 78), comparable to that in beating whole heart. Increasing intracellular Ca2+ did not significantly change NAD reduction. NAD reduction decreased reversibly to 11 +/- 1% (n = 78) in contracting myocytes electrically paced at 5 Hz for 10 min. Oxygen uptake was stimulated fivefold. There was minimal change in sarcoplasmic pH measured by fluorescence of carboxy-seminaphthorhodafluor-1. However, NAD reduction increased reversibly in response to electrically paced contractions when: (a) myoglobin was inactivated with sodium nitrite (37 +/- 7%; n = 48); or (b) cells were more densely layered and gassed with 20% O2/5% CO2 (48 +/- 3%; n = 30). We conclude that (a) the ratio NADH/NAD is decreased in well-oxygenated cells with increased work; (b) steady-state NAD reduction is increased with increased work when oxygen delivery is limited; and (c) functional myoglobin ensures an oxygen supply to the mitochondria of working cells.

[Study of nonequilibrium states of cyanide complexes of hemoglobin and myoglobin by a low-temperature inhibition method]. / Issledovanie neravnovesnykh sostoianii tsanidnykh kompleksov gemoglobina metodom nizkotemperaturnogo ingibirovaniia.

Cyanide-ferrihemoglobin (myoglobin) interaction was studied by freeze-quenching technique and low-temperature ESR at various pH values. New ESR signals of these low-spin cyanide-protein complexes were recorded. These signals were interpreted as indication of the appearance of conformationally non-equilibrium states of these complexes. The relaxation to the equilibrium states of corresponding proteins takes about hundred milliseconds.

Myoglobin-dependent oxidative metabolism in the hypoxic rat heart.

The role of myoglobin in facilitating O2 diffusion for oxidative energy production was investigated at high (0.9 mM) and low (0.1 mM) O2 tensions in the Langendorff-perfused rat heart. 31P nuclear magnetic resonance was used to monitor the intracellular pH and concentrations of high energy phosphates. NaNO2 or phenylhydrazine was used to inactivate greater than 85% of intracellular myoglobin. During hypoxia, ATP and phosphocreatine were depleted significantly more rapidly in hearts with reduced concentrations of functional myoglobin than in control hearts. However, at 0.9 mM O2, myoglobin inactivation did not limit oxidative energy metabolism. It is concluded that facilitation of O2 diffusion by cardiac myoglobin plays a significant role in O2 delivery to the mitochondria at low O2 tensions.

Inactivation of myoglobin by ortho-substituted arylhydrazines. Formation of prosthetic heme aryl-iron but not N-aryl adducts.

Stable phenyl-iron complexes are known to form in the reactions of myoglobin, hemoglobin, and catalase with phenylhydrazine. The phenyl moiety in these complexes migrates from the iron to a nitrogen of the porphyrin upon denaturation of the hemoproteins. Complexes obtained from myoglobin and ortho-substituted phenylhydrazines, however, are much less stable, have distinct chromophores, and do not yield N-arylporphyrins. These abnormal properties imply that the complexes differ in structure (e.g., they are aryldiazenyl-rather than aryl-iron complexes) or that ortho substitution strongly alters the chemistry of aryl-iron complexes. The present NMR studies unambiguously demonstrate that ortho-substituted phenylhydrazines give normal aryl-iron complexes but that the aryl group in these complexes is conformationally locked and is unable to shift from iron to nitrogen.