Pseudoperoxidase Activity Research Articles

Role of lactadherin in the clearance of phosphatidylserine‐expressing red blood cells

In red blood cells (RBCs) anionic phospholipids, such as phosphatidylserine, are present in the inner leaflet of the membrane bilayer. Exposure of phosphatidylserine occurs during senescence and during long-term storage of RBCs and is considered as the tag for removal from the circulation by macrophages. Lactadherin is a phosphatidylserine-binding glycoprotein secreted by macrophages that promotes the engulfment of phosphatidylserine-expressing apoptotic lymphocytes. This study investigates the role of lactadherin in the phagocytosis of phosphatidylserine-expressing RBCs. Transbilayer movement of phosphatidylserine was induced in RBCs either by storage beyond 30 days or by treatment with calcium ionophore A23187 and N-ethylmaleimide. Phosphatidylserine-expressing RBCs were incubated with phorbol ester-stimulated THP-1, and phagocytosis was determined by measuring the pseudoperoxidase activity of hemoglobin. The in vivo clearance of phosphatidylserine-enriched RBCs was measured in lactadherin-deficient mice and in their littermate controls. Lactadherin promoted phagocytosis of phosphatidylserine-expressing RBCs by macrophages in a concentration-dependent manner. Splenic macrophages from lactadherin-deficient mice had diminished capacity to phagocytose phosphatidylserine-expressing RBCs. The life span of RBCs in lactadherin-deficient mice was similar to wild-type littermate controls in vivo. However, when an excess of phosphatidylserine-expressing RBCs were infused, there was only a mild impairment in the clearance in lactadherin-deficient mice compared to wild-type littermate controls. These results show that clearance of phosphatidylserine-expressing RBCs is not diminished in a significant way in lactadherin-deficient mice under physiologic conditions and suggest the presence of redundant pathways.

The phosphatidylserine receptor mediates phagocytosis by vascular smooth muscle cells

Apoptosis participates in every step of atherogenesis, but the process of clearance of apoptotic cells by phagocytosis has been underestimated. Rapid removal of apoptotic cells is critical for tissue homeostasis, in order to avoid accumulation of necrotic material and subsequent inflammation in the pathological vascular wall. We have demonstrated by RT-PCR, western blot and immunocytofluorescence that vascular smooth muscle cells (VSMCs) express the phosphatidylserine receptor (PSR). We then tested the involvement of PSR in the ability of VSMCs to bind and engulf apoptotic cells. We used a model of senescent erythrocytes, which expose PS after 4 days of culture (85% of cells relative to 8% in freshly isolated erythrocytes). The pseudo-peroxidase activity of haemoglobin contained within erythrocytes allowed us to quantify per se both binding and phagocytosis by VSMCs. We have also shown by light and confocal microscopy that VSMCs were able to ingest aged erythrocytes. Addition of a blocking antibody or transfection of VSMCs by a siRNA directed against PSR reduced the binding and engulfment of aged erythrocytes by more than 90%. These results suggest that PSR is involved in phagocytosis of PS-presenting cells. Incubation of aged erythrocytes with VSMCs also significantly increased the expression of PSR, suggesting that the tethering/ingestion of apoptotic cells triggers this process. Immunostaining for PSR in complicated atherosclerotic plaques shows positivity in the media and macrophage-rich areas. The mechanisms underlying phagocytosis and involving PSR in vivo, within the pathological arterial wall, deserve further investigation.

Performance Characteristics and Comparison of Two Fecal Occult Blood Tests in Patients Undergoing Colonoscopy

We investigated the use of a new type of FOBT (EZ-Detect) that uses the blood's pseudo-peroxidase activity as an enzymatic catalyst, in a one-step chromogen-substrate system performed by the patient. Asymptomatic patients >/=50 years old received three Hemoccult II (HO) cards and three EZ-Detect (EZ) packages to be used in three consecutive bowel movements. Sensitivity, specificity, positive predictive value, and negative predictive value for detection of colorectal neoplasia was calculated. The study included 207 patients, with a mean age of 58.9 years. Diagnostic accuracy for detection of adenomas was similar for the EZ and HO tests (66.7% vs. 71.0%; P=0.48), while for advanced adenomas diagnostic accuracy for the EZ and HO tests was 86.0% vs. 94.2% (P=0.01), respectively. Most patients preferred the EZ test (92% vs. 8%). We conclude that the EZ test has a diagnostic profile similar to that of the HO test for identification of adenomas; however, for advanced adenomas the diagnostic accuracy was slightly better for the HO. The EZ test was preferred by most patients, which may increase colorectal cancer screening compliance.

Saliva plays a dual role in oxidation process in stomach medium

The aim of this study was to evaluate the role of saliva in the oxidation process under the acidic condition of the stomach. Saliva specimens played varied roles in the lipid peroxidation process of heated muscle tissue in simulated gastric fluid: pro-oxidant effects, no effects, and antioxidant effects. To elucidate these differences, selected saliva components were examined. The pseudoperoxidase activity of lactoperoxidase increased lipid peroxidation, while thiocyanate and nitrite-reduced lipid peroxidation. The effect of a saliva specimen on lipid peroxidation was correlated with the concentration of nitrite in the specimen, but not with that of other saliva components. The inhibitory effect of nitrite may be due to its conversion to NO. Elucidation of the antioxidant effect of saliva on co-oxidation of d-α-tocopherol in gastric fluid, demonstrated that saliva alone cannot protect d-α-tocopherol from co-oxidation, although it partially protected against lipid peroxidation. The presence of red wine polyphenols in stomach medium totally inhibits food lipid peroxidation and d-α-tocopherol co-oxidation.

Hydrogen-bonding conformations of tyrosine B10 tailor the hemeprotein reactivity of ferryl species

Ferryl compounds [Fe(IV)=O] in living organisms play an essential role in the radical catalytic cycle and degradation processes of hemeproteins. We studied the reactions between H2O2 and hemoglobin II (HbII) (GlnE7, TyrB10, PheCD1, PheE11), recombinant hemoglobin I (HbI) (GlnE7, PheB10, PheCD1, PheE11), and the HbI PheB10Tyr mutant of L. pectinata. We found that the tyrosine residue in the B10 position tailors, in two very distinct ways, the reactivity of the ferryl species, compounds I and II. First, increasing the reaction pH from 4.86 to 7.50, and then to 11.2, caused the the second-order rate constant for HbII to decrease from 141.60 to 77.78 M-1 s-1, and to 2.96 M-1 s-1, respectively. This pH dependence is associated with the disruption of the heme-tyrosine (603 nm) protein moiety, which controls the access of the H2O2 to the hemeprotein active center, thus regulating the formation of the ferryl species. Second, the presence of compound I was evident in the UV-vis spectra (648-nm band) in the reactions of HbI and recombinant HbI with H2O2, This band, however, is completely absent in the analogous reaction with HbII and the HbI PheB10Tyr mutant. Therefore, the existence of a hydrogen-bonding network between the heme pocket amino acids (i.e., TyrB10) and the ferryl compound I created a path much faster than 3.0x10(-2) s-1 for the decay of compound I to compound II. Furthermore, the decay of the heme ferryl compound I to compound II was independent of the proximal HisF8 trans-ligand strength. Thus, the pH dependence of the heme-tyrosine moiety complex determined the overall reaction rate of the oxidative reaction limiting the interaction with H2O2 at neutral pH. The hydrogen-bonding strength between the TyrB10 and the heme ferryl species suggests the presence of a cycle where the ferryl consumption by the ferric heme increases significantly the pseudoperoxidase activity of these hemeproteins.

Plasmodium falciparum: Discovery of peroxidase active organelles

Staining with 3,3′ diaminobenzidine tetrahydrochloride (DAB) is a common method used for the detection of peroxidases. Using this histochemical staining method in conjunction with transmission electron microscopy, we observed oxidation of DAB that was localized to a discrete set of organelles displaying morphological similarity to small (75–90 nm diameter) versions of higher eukaryotic microbodies or peroxisomes. These single membrane bounded organelles were characterized by an asymmetrical matrix capable of oxidizing DAB to an electron dense inclusion. Oxidation of DAB was further found to be dependent upon hydrogen peroxide (H 2O 2) as a substrate. Given a lack of peroxisomal import proteins and enzymes, it is unlikely that these represent conventional peroxisomes. Rather, they likely represent specialized organelles containing endogenous peroxidase or pseudo-peroxidase activity.

An improved colorimetric method for quantitation of heme using tetramethylbenzidine as substrate

Heme, an essential molecule with various biological functions, is often released from degraded hemoprotein including hemoglobin, cytochrome, hemopexin, peroxidase, and catalase. Quantitation of heme has been used in detection of malaria [1] and studies of heme interaction [2,3]. Current quantitative assays for heme require expensive instrumentation such as a spectroXuorometer [4], or the use of toxic substances such as pyridine [5]. In addition, several colorimetric methods have been developed using NaOH [6], sodium dodecyl sulfate [7] or Triton–methanol [8]. Among these colorimetric methods, Triton–methanol has been reported as the most sensitive method. Based on oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) by the pseudoperoxidase activity of heme [9], we describe here a simple colorimetric assay with a high sensitivity using 1-StepTM Turbo TMBELISA substrate (Turbo-TMB, Pierce No. 34022). We demonstrated that the Turbo-TMB assay is an easily performable method with a higher sensitivity than that of the Triton–methanol method. Heme was purchased from Sigma. A stock heme solution was prepared fresh by dissolving hemin chloride in 20mM NaOH and then centrifuging for 10min at 15,000 rpm to remove remaining hemin crystal. Heme concentrations were estimated from the absorbance at 385 nm using mMD58,400 in 100 mM NaOH [6], and adjusted to 1 mM by diluting with 20mM NaOH. All absorption spectra and absorbance were recorded on a Hitachi

Catalytic activity, stability, unfolding, and degradation pathways of engineered and reconstituted myoglobins

The structural and functional consequences of engineering a positively charged Lys residue and replacing the natural heme with a heme-L-His derivative in the active site of sperm whale myoglobin (Mb) have been investigated. The main structural change caused by the distal T67K mutation appears to be mobilization of the propionate-7 group. Reconstitution of wild-type and T67K Mb with heme-L-His relaxes the protein fragment around the heme because it involves the loss of the interaction of one of the propionate groups which stabilize heme binding to the protein. This modification increases the accessibility of exogenous ligands or substrates to the active site. The catalytic activity of the reconstituted proteins in peroxidase-type reactions is thus significantly increased, particularly with T67K Mb. The T67K mutation slightly reduces the thermodynamic stability and the chemical stability of Mb during catalysis, but somewhat more marked effects are observed by cofactor reconstitution. Hydrogen peroxide, in fact, induces pseudo-peroxidase activity but also promotes oxidative damage of the protein. The mechanism of protein degradation involves two pathways, which depend on the evolution of radical species generated on protein residues by the Mb active species and on the reactivity of phenoxy radicals produced during turnover. Both protein oligomers and heme-protein cross-links have been detected upon inactivation.

Transferrin enzyme immunoassay for quantitative monitoring of blood contamination in saliva.

When blood components are present in the oral mucosa, quantitative estimates of salivary hormone concentrations may be compromised (1). Blood and its components can leak into the oral mucosa as a result of micro injuries such as burns or abrasions. The probability of blood leakage is increased with poor oral health (i.e., periodontal disease), certain infectious diseases (e.g., HIV) (2), and behavior known to influence oral health negatively (e.g., smoking) (3). Few studies, however, have investigated methods to determine blood contamination in saliva. The Hemastix® Reagent Strip for Urinalysis has been used to test for blood in saliva (4)(5). This method detects the pseudoperoxidase activity of hemoglobin; thus, endogenous salivary peroxidases produce false-positive results for hemoglobin. To provide a quantitative measure of blood contamination in saliva, we developed an enzyme immunoassay for transferrin, which is present in very small amounts ( 1000 mg/L). We expect that serum and salivary concentrations of transferrin are not correlated and that transferrin, with a formula weight of 76 000 and radius of 5 nm, would reach saliva in high concentrations only when the barrier between blood and saliva is compromised. Saliva (by passive drool) and serum samples were collected without stimulation simultaneously from 40 young adults. The Pennsylvania State University Institutional Review Board approved all procedures, and informed consent was obtained. All samples were stored at −40 °C. All samples were thawed, vortex-mixed, and centrifuged at 1500 g for 15 min before assay for serum transferrin, salivary transferrin, or testing by Hemastix Reagent …

Oxidative Degradation of Cardiotoxic Anticancer Anthracyclines to Phthalic Acids: NOVEL FUNCTION FOR FERRYLMYOGLOBIN

We show that the pseudoperoxidase activity of ferrylmyoglobin (MbIV) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. MbIV, formed in vitro by reacting horse heart MbIII with H2O2, caused disappearance of the spectrum of DOX at 477 nm and appearance of UV-absorbing chromophores that indicated opening and degradation of its tetracyclic ring. Electron spray ionization mass spectrometry analyses of DOX/MbIV ultrafiltrates showed that DOX degradation resulted in formation of 3-methoxyphthalic acid, the product of oxidative modifications of its methoxy-substituted ring D. Other methoxy-substituted anthracyclines similarly released 3-methoxyphthalic acid after oxidation by MbIV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and MbIII/H2O2 or hemin/H2O2 showed that the porphyrin radical of MbIV-compound I and the iron-oxo moiety of MbIV-compound II were sequentially involved in oxidizing DOX; however, oxidation by compound I formed more 3-methoxyphthalic acid than oxidation by compound II. Sizeable amounts of 3-methoxyphthalic acid were formed in the heart of mice treated with DOX, in human myocardial biopsies exposed to DOX in vitro, and in human cardiac cytosol that oxidized DOX after activation of its endogenous myoglobin by H2O2. Importantly, H9c2 cardiomyocytes were damaged by low concentrations of DOX but could tolerate concentrations of 3-methoxyphthalic acid higher than those measured in murine or human myocardium. These results unravel a novel function for MbIV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity.

Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity

Studying the effects of microgravity on cell differentiation will enhance our understanding of fundamental biology and is indispensable for a sustained space program. Rauscher murine erythroleukemic cells were chosen as a model system to study erythroid cell differentiation aboard the International Space Station because these cells undergo differentiation in response to the natural inducer, erythropoietin, as well as various chemical-inducers. We have now developed a method to quantify hemoglobin in Rauscher cells after weeks of fixation and storage required for such space biology experiments. By exploiting the pseudoperoxidase activity of hemoglobin and by using reagents that yield a soluble chromophore that freely passes out of fixed cells, we developed a highly specific and sensitive assay applicable to cells fixed as long as 4 months.

Pseudoperoxidase activity of myoglobin: kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates.

Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.

Monoamine-dependent production of reactive oxygen species catalyzed by pseudoperoxidase activity of human hemoglobin.

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, beta-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O2*-). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc'). The O2*- generation and Asc formation were detected by O2*--specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O2*- production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H2O2, but a trace of H2O2 added to the system greatly increased the production of both O2*- and Asc*. Addition of GSH completely inhibited the PEA-dependent production of O2*- and Asc* in Hb solution. We propose that the O2*- generation and Asc* formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.

Hemoglobin can nitrate itself and other proteins

Incubation of human hemoglobin with nitrite and hydrogen peroxide was found to induce autonitration and nitration of another protein (bovine serum albumin), as demonstrated by detection of nitrotyrosine residues in Western blots of separated membrane proteins. Inhibition of nitration by conversion of hemoglobin into the cyanmet form demonstrates that nitration is due to the pseudoperoxidase activity of hemoglobin. Incubation of whole erythrocytes with nitrite and hydrogen peroxide induces nitration of erythrocyte membrane proteins, much stronger when cellular catalase was inhibited with azide. These results suggest that hemoglobin and other hemoproteins may contribute to the tyrosine nitration in vivo.

Redox reactions of hemoglobin and myoglobin: biological and toxicological implications.

Direct cytotoxic effects associated with hemoglobin (Hb) or myoglobin (Mb) have been ascribed to redox reactions (involving either one- or two-electron steps) between the heme group and peroxides. These interactions are the basis of the pseudoperoxidase activity of these hemoproteins and can be cytotoxic when reactive species are formed at relatively high concentrations during inflammation and typically lead to cell death. Peroxides relevant to biological systems include hydrogen peroxide, lipid hydroperoxides, and peroxynitrite. Reactions between Hb/Mb and peroxides form the ferryl oxidation state of the protein, analogous to compounds I and II formed in the catalytic cycle of many peroxidase enzymes. This higher oxidation state of the protein is a potent oxidant capable of promoting oxidative damage to most classes of biological molecules. Free iron, released from Hb, also has the potential to promote oxidative damage via classical "Fenton" chemistry. It has become increasingly evident that Hb/Mb redox reactions or their by-products play a critical role in the pathophysiology of some disease states. This review briefly discusses the reactions of Hb/Mb with biological peroxides, potential cytotoxicity and the impact of these interactions on modulation of cell signaling pathways regulated by these reactive species. Also discussed in this article is the role of heme-protein chemistry in relation to the toxicity of hemoproteins.

Oxidative mechanisms of hemoglobin-based blood substitutes.

Chemically or genetically altered cell-free hemoglobin (Hb) has been developed as an oxygen carrying therapeutic. Site-directed modifications are introduced and serve to stabilize the protein molecules in a tetrameric and/or a polymeric functional form. Direct cytotoxic effects associated with cell-free Hb have been ascribed to redox reactions (involving either 1 or 2 electron steps) between the heme group and peroxides. These interactions are the basis of the pseudoperoxidase activity of Hb and can be cytotoxic when reactive species are formed at relatively high concentrations during inflammation and typically lead to cell death. Peroxides relevant to biological systems include hydrogen peroxide (H2O2), lipid hydroperoxides (LOOH), and peroxynitrite (ONOO-). Reactions between Hb and peroxides form the ferryl oxidation state of the protein, analogous to compounds I and II formed in the catalytic cycle of many peroxidase enzymes. This higher oxidation state of the protein is a potent oxidant capable of promoting oxidative damage to most classes of biological molecules. Further complications are thought to arise through the disruption of key signaling pathways resulting from alteration of or destruction of important physiological mediators.

Mechanism of inhibition of porcine leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.

The mechanism of inhibition of porcine leukocyte 12-lipoxygenase by 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was investigated. This compound is selective for the leukocyte form of the 12-lipoxygenase and inhibits the purified recombinant enzyme with an IC(50) value of approximately 2 microM. OPP induced a concentration-dependent lag phase in the oxygenation of arachidonic acid and decreased the maximal rate of reaction. Addition of the fatty acid hydroperoxide 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE) to the reaction greatly reduced the OPP-induced lag. Lineweaver-Burk analysis of the effect of OPP on 12-lipoxygenase kinetics with arachidonic acid indicated that it was a mixed-type inhibitor. OPP was not metabolized by 12-lipoxygenase as evidenced by its quantitative recovery from incubations with stoichiometric amounts of enzyme and 13-HPODE or arachidonic acid. OPP inhibited the pseudoperoxidase activity of the enzyme with 13-HPODE and the reducing agent, BWA137C. Lineweaver-Burk analysis of the effect of OPP on pseudoperoxidase kinetics suggested that OPP was competitive with 13-HPODE. Single-turnover experiments indicated that OPP inhibited the reduction of 13-HPODE by a stoichiometric amount of ferrous 12-lipoxygenase. Addition of 13-HPODE shortened the OPP-induced lag phase but did not affect the maximal rate of enzyme activity. In addition, OPP had no effect on total product formation in either the presence or the absence of 5 microM 13-HPODE when the reaction was allowed to go to completion. All of these observations are consistent with a model for inhibition of 12-lipoxygenase activity in which OPP slows the oxidation of the inactive ferrous enzyme to the active ferric enzyme and competes with arachidonic acid for the ferric enzyme.

COMPARISON OF SCREENING METHODS IN THE DETECTION OF BLADDER CANCER

We prospectively evaluate and compare the sensitivity and specificity of urine cytology, BTA stat, NMP22, fibrin/fibrinogen degradation products (FDP), telomerase, chemiluminescent hemoglobin and hemoglobin dipstick to detect bladder cancer. Single voided specimens were obtained from 57 patients with bladder cancer, and 139 without evidence of bladder malignancy on cystoscopy or a negative biopsy of indeterminate lesions. A cytology report was available for 125 patients and interpreted independently. BTA stat, NMP22 and FDP were analyzed according to manufacturer specifications. The telomerase assay was performed on cells collected from urine by centrifugation in preparation for polymerase chain reaction based amplification using the telomeric repeat amplification protocol assay. The chemiluminescent screening assay for hemoglobin in urine uses the pseudoperoxidase activity of hemoglobin on hydrogen peroxide and subsequent oxidation of 7-dimethylaminonaphthalene-1,2-dicarbonic acid hydrazide to generate chemiluminescence emission. Hemoglobin dipstick was interpreted as positive if the hemoglobin content in the urine was trace or greater. Overall sensitivity with urine cytology, BTA stat, NMP22, FDP, telomerase, chemiluminescent hemoglobin and the hemoglobin dipstick was 44, 74, 53, 52, 70, 67 and 47%, respectively. Specificity with cytology, telomerase and FDP was high (95, 99 and 91%, respectively) but BTA stat, NMP22 (optimized), chemiluminescent hemoglobin (optimized) and the hemoglobin dipstick demonstrated lower specificity of 73, 60, 63 and 84%, respectively. Stepwise logistic regression analysis revealed that for all tumors, and within each tumor grade and stage telomerase had the strongest association with bladder cancer among all tests (69% overall concordance). Telomerase was also positive in 91% of the patients (10 of 11) with carcinoma in situ. Urinary telomerase had the highest combination of sensitivity and specificity (70 and 99%, respectively) for bladder cancer screening in these patients. It was the strongest predictor with superior accuracy in patients with grade 1 and noninvasive tumors (pTa), and extremely useful in patients with carcinoma in situ. Telomerase appears to be promising and outperformed cytology, BTA stat, NMP22, FDP, chemiluminescent hemoglobin and hemoglobin dipstick in the prediction of bladder cancer.

Reactions of Sperm Whale Myoglobin with Hydrogen Peroxide: EFFECTS OF DISTAL POCKET MUTATIONS ON THE FORMATION AND STABILITY OF THE FERRYL INTERMEDIATE

Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37 degreesC. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy- to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60% increase in the initial oxidation reaction and a much larger 2. 5-fold increase in the rate of autoreduction. Double insertion of Phe at both the B10- and E11-positions (L29F/V68F) produces a mutant with oxidation characteristics of both single mutants, slow initial oxidation, and rapid autoreduction, but an extraordinarily high affinity for O2. Replacing His-64(E7) with Gln produces 3-4-fold increases in both processes. Combining the mutation H64Q with L29F results in a myoglobin with enhanced resistance to metMb formation in the absence of antioxidant enzymes (i.e. catalase and superoxide dismutase) due to its own high pseudoperoxidase activity, which rapidly removes any HOOH produced in the initial stages of autoxidation. This double substitution occurs naturally in the myoglobin of Asian elephants, and similar multiple replacements have been used to reduce selectively the rate of nitric oxide (NO)-induced oxidation of both recombinant MbO2 and HbO2 blood substitute prototypes without altering O2 affinity.

A colorimetric–enzymatic microassay for the quantitation of antibody-dependent complement activation

In this report we describe a reliable, sensitive, safe, and easy way to assess antibody-dependent complement-mediated hemolysis. The assay is based on the quantitation of hemoglobin (Hb) released from lysed erythrocytes indirectly, through the generation of fluorene blue, a compound formed from 2-7 diaminofluorene in an enzymatic reaction catalyzed by the Hb molecule. The fact that Hb is the most abundant protein within a mature RBC (∼10 11 molecules per cell) and possesses pseudoperoxidase activity, makes the fluorene blue-coupled assay more sensitive than the simple estimation of Hb adsorption at 410 nm (Soret adsorption maxima of Hb), and as sensitive and reliable as its radioactive equivalent based on the release of 51 Cr from previously loaded RBCs. Using this assay chimeric mouse–human anti-dansyl antibodies, comprising all the human IgG isotypes, were tested for their ability to mediate complement activation. The results obtained agreed with previously reported data, confirming that the fluorene blue-coupled assay is reliable. This assay also has significant advantages over the radioactive-based assay in that the reagents used are inexpensive, and the concerns of using radioactivity and the associated hazards are obviated. Because there is no need for loading the target cells with the analyte to be detected since RBCs are already loaded with Hb, the fluorene blue-coupled assay is simpler and eliminates many steps in comparison to the 51 Cr release based assay.