Oxidation of anti-thyroid drugs and their selenium analogs by ABTS radical cation.

Lactoperoxidase was previously used as a model enzyme to test the inhibitory activity of selenium analogs of anti-thyroid drugs with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Peroxidases oxidize ABTS to a metastable radical ABTS•+, which is readily reduced by many antioxidants, including thiol-containing compounds, and it has been used for decades to measure antioxidant activity in biological samples. We showed that anti-thyroid drugs 6-n-propyl-2-thiouracil, methimazole, and selenium analogs of methimazole also reduced it rapidly. This reaction may explain the anti-thyroid action of many other compounds, particularly natural antioxidants, which may reduce the oxidized form of iodine and/or tyrosyl radicals generated by thyroid peroxidase thus decreasing the production of thyroid hormones. However, influence of selenium analogs of methimazole on the rate of hydrogen peroxide consumption during oxidation of ABTS by lactoperoxidase was moderate. Direct hydrogen peroxide reduction, proposed before as their mechanism of action, cannot therefore account for the observed inhibitory effects. 1-Methylimidazole-2-selone and its diselenide were oxidized by ABTS•+ to relatively stable seleninic acid, which decomposed slowly to selenite and 1-methylimidazole. In contrast, oxidation of 1,3-dimethylimidazole-2-selone gave selenite and 1,3-dimethylimidazolium cation. Accumulation of the corresponding seleninic acid was not observed.

Structure of a ternary complex of lactoperoxidase with iodide and hydrogen peroxide at 1.77 Å resolution.

Lactoperoxidase (LPO) is a mammalian heme peroxidase which catalyzes the conversion of thiocyanate (SCN¯) and iodide (I-) by hydrogen peroxide (H2O2) into antimicrobial hypothiocyanite (OSCN¯) and hypoiodite (IO-). The prosthetic heme group is covalently attached to LPO through two ester linkages involving conserved glutamate and aspartate residues. On the proximal side, His351 is coordinated to heme iron while His 109 is located in the substrate binding site on the distal heme side. We report here the first structure of the ternary complex of LPO with iodide (I-) and H2O2 at 1.77 Å resolution. LPO was crystallized with ammonium iodide and the crystals were soaked in the reservoir solution containing H2O2. Structure determination showed the presence of an iodide ion and a H2O2 molecule in the substrate binding site. The iodide ion occupied the position which is stabilized by the interactions with heme moiety, His109, Arg255 and Glu258 while H2O2 was held between the heme iron and His109. The presence of I- in the distal heme cavity seems to screen the positive charge of Arg255 thus suppressing the proton transfer from H2O2 to His109. This prevents compound I formation and allows trapping of a stable enzyme-substrate (LPO-I--H2O2) ternary complex. This stable geometrical arrangement of H2O2 in the distal heme cavity of LPO is similar to that of H2O2 in the structure of the transient intermediate of the palm tree heme peroxidase. The biochemical studies showed that the catalytic activity of LPO decreased when the samples of LPO were preincubated with ammonium iodide.

Potassium-induced partial inhibition of lactoperoxidase: structure of the complex of lactoperoxidase with potassium ion at 2.20 Å resolution.

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

Structure of Yak Lactoperoxidase at 1.55 Å Resolution.

Lactoperoxidase (LPO) is a heme containing oxido-reductase enzyme. It is secreted from mammary, salivary, lachrymal and mucosal glands. It catalyses the conversion of thiocyanate into hypothiocyanate and halides into hypohalides. LPO belongs to the superfamily of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The heme prosthetic group is covalently linked in LPO through two ester bonds involving conserved residues Glu258 and Asp108. It was isolated from colostrum of yak (Bos grunniens), purified to homogeneity and crystallized using ammonium iodide as a precipitating agent. The crystals belonged to monoclinic space group P21 with cell dimensions of a = 53.91 Å, b = 78.98 Å, c = 67.82 Å and ß = 92.96°. The structure was determined at 1.55 Å resolution. This is the first structure of LPO from yak. Also, this is the highest resolution structure of LPO determined so far from any source. The structure determination revealed that three segments (Ser1-Cys15), (Thr117-Asn138) and (Cys167-Leu175) were disordered and formed one surface of LPO structure. In the substrate binding site, the iodide ions were observed in three subsites which are formed by (1) heme moiety and residues, Gln105, Asp108, His109, Phe113, Arg255, Glu258, Phe380 and Phe381, (2) residues, Asn230, Lys232, Pro236, Cys248, Phe254, Phe381 and Pro424 and (3) residues, Ser198, Leu199 and Arg202. The structure determination also revealed that the side chain of Phe254 was disordered. It was observed to adopt two conformations in the structures of LPO.

Effects of oral administration of colostrum whey in peripartum goat on antimicrobial peptides in postpartum milk.

The present study was conducted to examine whether colostrum supplementation in peripartum goats increases the antimicrobial peptides in their milk. Goats were orally administered 2 ml of colostrum whey products (colostrum group) or water (control group) daily, from 2 weeks before until 2 weeks after kidding. Body weights of mothers and kids were measured. Blood, milk, and fecal samples were collected from the mothers, and blood samples were collected from the kids. Concentrations of milk antimicrobial peptides (beta-defensin, cathelicidin, lactoferrin, S100A7, lactoperoxidase, and immunoglobulin A [IgA]) were determined. IgA and nutritional parameters (glucose, total cholesterol, triglyceride, ketone bodies, and non-esterified fatty acids) were also determined in the blood of mothers and kids. Milk IgA and lactoferrin concentrations were higher in the colostrum group than in the control group. Conversely, lower milk concentrations of S100A7 were observed in the colostrum group than that in the control group. Plasma IgA concentrations were higher for kids from the colostrum group than for those from the control group. These results suggest that oral administration of colostrum in pregnant goats increases IgA concentration in postpartum milk, which can subsequently improve the health of their kids.

Long-term chromium picolinate supplementation improves colostrum profile of Santa Ines ewe.

Chromium (Cr) is a micromineral that is involved in the metabolism of carbohydrates, lipids, ammonia, and nucleic acids; thus, its supplementation can influence the nutritional status of ruminants, and consequently, colostrum profile, since this secretion depends on products secreted by the mammary gland and elements of the maternal bloodstream. The present study investigated the influence of supplementation with Cr bound to organic molecule on the nutritional, immune, and antioxidant quality of ewe colostrum. Thirty-two multiparous Santa Ines ewes (55.3 ± 8.00 kg body weight) were randomly assigned into four groups: T1 (0.0 mg of chromium picolinate (CrPic) supplementation per ewe, n = 8), T2 (0.15 mg of CrPic per ewe, n = 9), T3 (0.30 mg of CrPic per ewe, n = 7), and T4 (0.45 mg of CrPic per ewe, n = 8). Supplementation was supplied during the breeding season, pregnancy, and lactation. Shortly after calving, the first milking colostrum was collected to determine its chemical composition, activity of lysozyme, lactoperoxidase, ceruloplasmin, catalase, glutathione peroxidase, and oxygen radical absorbance capacity. The results show that lactoperoxidase activity decreased with CrPic supplementation (P < 0.01), revealing that this micromineral reduces an important component of defense mechanism in the body. Therefore, the results of this work show that supplementation with chromium picolinate influences colostrum quality.

Reactivation of peroxidase activity in human saliva samples by polyphenols.

OBJECTIVES: The enzyme lactoperoxidase (LPO), which is released into several body fluids like saliva, is an essential part to maintain the oral bacterial homeostasis by catalysing the oxidation of thiocyanate (SCN-) to hypo-thiocyanite (-OSCN). The formation of unreactive redox intermediates (like Compound II) leads to a decreased pseudo-halogenating enzyme activity, which is associated with a higher risk for oral infections. According to former studies with bovine LPO selected flavonoids were tested in respect to their potential to reactivate the enzymatic activity in a more physiological, human salivary system. DESIGN: Saliva samples from healthy donors were collected and characterized by using several gel staining methods and immunoblotting. Afterwards kinetic measurements were performed by applying the TNB-assay to evaluate the pseudo-halogenating salivary peroxidase (SAPX) activity. The measurements were performed in the presence of excess H2O2 to simulate pro-inflammatory conditions. Moreover selected flavonoids or an ethanolic extract of Tormentillae rhizoma were applied to test their regenerating effect on the LPO-derived -OSCN production. RESULTS: Despite the complex protein composition of the collected saliva samples, an SAPX-derived pseudo-halogenating activity could be identified. The -OSCN regenerating effects of the tested polyphenols were completely comparable to previous in vitro experiments with bovine LPO. Thus, we could show that phenolic substances are suitable to regenerate the peroxidase activity in human saliva samples after H2O2-induced inactivation. CONCLUSION: The studies provide new insights into the effect of pharmaceutical relevant polyphenols on salivary peroxidase activity and thus, suggest this enzyme as a new target for the prevention and therapy of oral inflammatory diseases.

Tannins and Tannin-Related Derivatives Enhance the (Pseudo-)Halogenating Activity of Lactoperoxidase.

Several hydrolyzable tannins, proanthocyanidins, tannin derivatives, and a tannin-rich plant extract of tormentil rhizome were tested for their potential to regenerate the (pseudo-)halogenating activity, i.e., the oxidation of SCN- to hypothiocyanite -OSCN, of lactoperoxidase (LPO) after hydrogen peroxide-mediated enzyme inactivation. Measurements were performed using 5-thio-2-nitrobenzoic acid in the presence of tannins and related substances in order to determine kinetic parameters and to trace the LPO-mediated -OSCN formation. The results were combined with docking studies and molecular orbital analysis. The -OSCN-regenerating effect of tannin derivatives relates well with their binding properties toward LPO as well as their occupied molecular orbitals. Especially simple compounds like ellagic acid or methyl gallate and the complex plant extract were found as potent enzyme-regenerating compounds. As the (pseudo-)halogenating activity of LPO contributes to the maintenance of oral bacterial homeostasis, the results provide new insights into the antibacterial mode of action of tannins and related compounds. Furthermore, chemical properties of the tested compounds that are important for efficient enzyme-substrate interaction and regeneration of the -OSCN formation by LPO were identified.

Set-up and application of an analytical approach for the quality control of purified colostrum as food supplement.

A validated analytical procedure is here described for the quality control of the protein fraction of purified bovine colostrum used in food supplements. The proposed procedure starts with 1D and 2D-gel electrophoresis. The sample is then separated into two fractions by protein G affinity chromatography: the IgG enriched and the IgG depleted fraction (IgG-d). A size exclusion chromatography coupled to UV is then applied to the IgG and IgG-d fractions for the quantitative analysis of IgG and IgM, respectively. The IgG-d fraction is then analysed by HPLC-MS analysis for the quantitative analysis of ß-lactoglobulins and α-lactoalbumin. The next step is to quantitatively measure a set of bioactive proteins selected from the bovine colostrum data bank on the basis of their claimed health benefits. The enzymatic activities of lactoperoxidase and xanthine dehydrogenase/oxidase are then tested as an index of protein functionality.

Components of a standardised olive leaf dry extract (Ph. Eur.) promote hypothiocyanite production by lactoperoxidase.

We investigated in vitro the ability of a standardised olive leaf dry extract (Ph. Eur.) (OLE) as well as of its single components to circumvent the hydrogen peroxide-induced inhibition of the hypothiocyanite-producing activity of lactoperoxidase (LPO). The rate of hypothiocyanite (⁻OSCN) formation by LPO was quantified by spectrophotometric detection of the oxidation of 5-thio-2-nitrobenzoic acid (TNB). By using excess hydrogen peroxide, we forced the accumulation of inactive enzymatic intermediates which are unable to promote the two-electronic oxidation of thiocyanate. Both OLE and certain extract components showed a strong LPO-reactivating effect. Thereby an o-hydroxyphenolic moiety emerged to be essential for a good reactivity with the inactive LPO redox states. This basic moiety is found in the main OLE components oleuropein, oleacein, hydroxytyrosol, caffeic acid as well as in different other constituents including the OLE flavone luteolin. As LPO is a key player in the humoral immune response, these results propose a new mode of action regarding the well-known bacteriostatic and anti-inflammatory properties of the leaf extract of Olea europaea L.

Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity.

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN(-)), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN(-) yields the bactericidal hypothiocyanite (OSCN(-)) in airway surface liquid (ASL). Although SCN(-) is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I(-)) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN(-), inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I(-) concentration, and this I(-) concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses.

Antineoplastic effect of iodine and iodide in dimethylbenz[a]anthracene-induced mammary tumors: association between lactoperoxidase and estrogen-adduct production.

Several groups, including ours, have reported that iodine exhibited antiproliferative and apoptotic effects in various cancer cells only if this element is supplemented as molecular iodine, or as iodide, to cells that are able to oxidize it with the enzyme thyroperoxidase. In this study, we analyzed the effect of various concentrations of iodine and/or iodide in the dimethylbenz[a]anthracene (DMBA) mammary cancer model in rats. The results show that 0.1% iodine or iodide increases the expression of peroxisome proliferator-activated receptor type γ (PPARγ), triggering caspase-mediated apoptosis pathways in damaged mammary tissue (DMBA-treated mammary gland) as well as in frank mammary tumors, but not in normal mammary gland. DMBA treatment induces the expression of lactoperoxidase, which participates in the antineoplastic effect of iodide and could be involved in the pro-neoplastic effect of estrogens, increasing the formation of DNA adducts. In conclusion, our results show that a supplement of 0.1% molecular iodine/potassium iodide (0.05/0.05%) exert antineoplastic effects, preventing estrogen-induced DNA adducts and inducing apoptosis through PPARγ/caspases in pre-cancer and cancerous cells. Since this iodine concentration does not modify the cytology (histology, apoptosis rate) or physiology (triiodothyronine and thyrotropin) of the thyroid gland, we propose that it be considered as an adjuvant treatment for premenopausal mammary cancer.

Invited review: Annatto usage and bleaching in dairy foods.

Annatto is a yellow/orange colorant that is widely used in the food industry, particularly in the dairy industry. Annatto, consisting of the carotenoids bixin and norbixin, is most commonly added to produce orange cheese, such as Cheddar, to achieve a consistent color over seasonal changes. This colorant is not all retained in the cheese, and thus a percentage remains in the whey, which is highly undesirable. As a result, whey is often bleached. Hydrogen peroxide and benzoyl peroxide are the 2 bleaching agents currently approved for bleaching whey in the United States. Recent studies have highlighted the negative effect of bleaching on whey flavor while concurrently there is a dearth of current studies on bleaching conditions and efficacy. Recent international mandates have placed additional concern on the use of benzoyl peroxide as a bleaching agent. This review discusses the advantages, disadvantages, regulatory concerns, flavor implications, and optimal usage conditions of 2 widely used bleaching agents, hydrogen peroxide and benzoyl peroxide, as well as a few alternative methods including lipoxygenase, peroxidase, and lactoperoxidase systems.

Challenge testing the lactoperoxidase system against a range of bacteria using different activation agents.

Lactoperoxidase (LP) exerts antimicrobial effects in combination with H(2)O(2) and either thiocyanate (SCN(-)) or a halide (e.g., I(-)). Garlic extract in the presence of ethanol has also been used to activate the LP system. This study aimed to determine the effects of 3 LP activation systems (LP+SCN(-)+H(2)O(2); LP+I(-)+H(2)O(2); LP + garlic extract + ethanol) on the growth and activity of 3 test organisms (Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus). Sterilized milk was used as the reaction medium, and the growth pattern of the organisms and a range of keeping quality (KQ) indicators (pH, titratable acidity, ethanol stability, clot on boiling) were monitored during storage at the respective optimum growth temperature for each organism. The LP+I(-)+ H(2)O(2) system reduced bacterial counts below the detection limit shortly after treatment for all 3 organisms, and no bacteria could be detected for the duration of the experiment (35 to 55 h). The KQ data confirmed that the milk remained unspoiled at the end of the experiments. The LP + garlic extract + ethanol system, on the other hand, had no effect on the growth or KQ with P. aeruginosa, but showed a small retardation of growth of the other 2 organisms, accompanied by small increases (5 to 10 h) in KQ. The effects of the LP+SCN(-)+H(2)O(2) system were intermediate between those of the other 2 systems and differed between organisms. With P. aeruginosa, the system exerted total inhibition within 10 h of incubation, but the bacteria regained viability after a further 5 h, following a logarithmic growth curve. This was reflected in the KQ indicators, which implied an extension of 15 h. With the other 2 bacterial species, LP+SCN(-)+H(2)O(2) exerted an obvious inhibitory effect, giving a lag phase in the growth curve of 5 to 10 h and KQ extension of 10 to 15 h. When used in combination, I(-) and SCN(-) displayed negative synergy.

Physical and sensory characteristics of marinated broiler drumsticks treated with lactoperoxidase system and thermal treatment.

1. The lactoperoxidase system (LPS) and thermal treatments have been shown to inactivate some micro-organisms in foods. However, further studies are needed to evaluate whether these treatments influenced the physical and sensory characteristics of treated samples. 2. A solution that contained 1% acetic acid and 3% salt with pH adjusted to 4 was developed as a standard marinade. The LPS consisting of 1 microg/ml lactoperoxidase (LP), 5.9 mM potassium thiocyanate (KSCN) and 2.5 mM hydrogen peroxide (H2O2) was added to the marinade for the LPS treatments. 3. In the thermal treatment, samples were heated with the marinade solution at 58 degrees C for 2 min and then marinated at 4 degrees C for 18 h, while the non-thermal treatments were marinated at 4 degrees C for 18 h. 4. For sensory evaluation, flavouring agents including 0.3% black pepper and 0.15% garlic powder were added to the marinade. For physical evaluation, no flavouring agents were added. 5. The results showed that combined LPS and thermal treatment did not impair the physical or sensory qualities of the samples. 6. In conclusion, marinated broiler drumsticks treated with LPS and thermal treatment had acceptable physical and sensory qualities.

[Therapeutic properties of proteins and peptides from colostrum and milk]. / Wlasciwosci terapeutyczne bialek i peptydów z siary i mleka.

Colostrum and milk are rich in proteins and peptides which play a crucial role in innate immunity when transferred to the offspring and may accelerate maturation of the immune system in neonates. The immunotropic properties of these proteins prompted investigators research their potential application in prevention and therapy. Lactoferrin (LF) exhibits antibacterial, antifungal, antiviral, antiparasitice, and antitumoral activities. It is protective with regard to intestinal epithelium, promotes bone growth, and accelerates the recovery of immune system function in immunocompromised animals. LF was tried in the treatment of hepatitis C infection and the intestinal form of graft-versus-host disease (GvHD). A proline-rich polypeptide (PRP) demonstrated a variety of immunotropic functions, including the promotion of T-cell maturation and inhibition of autoimmune disorders. PRP, in the form of chewable tablets (Colostrinin) was recently found to improve or stabilize the health status of Alzheimer's disease patients. Casein and casein-derived peptides showed protective activities in enamel demineralization and as caries-preventing agents. The protein hydrolyzates were also protective in diabetic animals, reduced tumor growth, had antihypertensive activity and diminished colicky symptoms in infants. Glycomacropeptide (GMP), a peptide derived from kappa-casein, exhibited various antibacterial and antithrombotic activities. Alpha-lactalbumin (LA) demonstrated antiviral, antitumoral and anti-stress properties. LA-enriched diets were anxiolytic, lowered blood pressure in rats, prevented diarrhea, and led to a better weight gain in malnourished children. HAMLET, a complex of LA and oleic acid, was effective in patients with cutaneous papillomas. Lysozyme found application in infant formulas, the treatment of periodentitis, and the prevention of tooth decay. Milk enriched in lysozyme was used in feeding premature infants suffering from concomitant diseases. Interesting, antibacterial properties were exhibited by lactoperoxidase. Both lysozyme and lactoperoxidase required cooperative action with LF in combating bacteria. In conclusion, preparations derived from milk and colostrum are effective, easily bioaccessible, and safe, finding wide application in prevention and therapy for newborns and adults.

Glutathione-dependent oxidative modification of protoporphyrin and other dicarboxylic porphyrins by mammalian and plant peroxidases.

Protoporphyrin, an intermediate in heme and chlorophyll biosynthesis, can accumulate in human and plant tissues under certain pathological conditions and is a photosensitizer used in cancer phototherapy. We previously showed that protoporphyrin and the related non-natural dicarboxylic porphyrin deuteroporphyrin are rapidly oxidized by horseradish peroxidase in the presence of some thiols, especially glutathione. This study reports that bovine lactoperoxidase, but not leucocyte myeloperoxidase, can also catalyze this reaction and that Tween and ascorbic acid are inhibitors. Exogenous hydrogen peroxide is not required and cannot replace glutathione. Deuteroporphyrin was oxidized to a unique green chlorin product with two oxygen functions added directly to the characteristic reduced pyrrole ring of the chlorin. Spectroscopic and chromatographic results suggest that protoporphyrin was oxidized not to a green chlorin, but to a much more polar red porphyrin modified by oxidative addition to the two vinyl side chains. Two related nonnatural dicarboxylic porphyrins, with ethyl or hydroxyethyl instead of vinyl side chains, are not substrates or products for this enzymatic conversion.

Expression of a functional thyroglobulin fragment in a baculovirus system.

The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3' end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel-nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.

Reactive nitrogen intermediates and antimicrobial activity: role of nitrite.

The reactive nitrogen intermediate (RNI) nitric oxide (NO.) is formed from L-arginine by an NO. synthase and, following secondary reactions yielding additional toxic intermediates, nitrite (NO2-) and nitrate are formed. Nitrite, however, also has toxic properties. At acid pH, nitrous acid (HNO2) is bactericidal to Escherichia coli, in association with the loss of HNO2/NO2- and the uptake of oxygen, an effect which is increased by H2O2. Under conditions in which HNO2/NO2- +/- H2O2 were ineffective, the further addition of peroxidase (myeloperoxidase [MPO], eosinophil peroxidase, lactoperoxidase) or catalase resulted in bactericidal activity and the disappearance of HNO2/NO2-. Paradoxically, HNO2/NO2- also inhibited the bactericidal activity of MPO by the formation of a complex with MPO with a shift in the absorption spectrum, and by reaction with hypochlorous acid (HOCl) (the product of the chloride-supplemented MPO-H2O2 system), with loss of the bactericidal activity of HOCl and the disappearance of both HOCl and HNO2/NO2- from the reaction mixture. Thus, HNO2/NO2-, rather than being solely an end product of RNI formation, may influence antimicrobial activity either by acting alone, with H2O2, or with H2O2 and peroxidase as a source of toxic agents, or by inhibiting the peroxidase-mediated antimicrobial systems.

Interacting properties of bovine lactoferrin with immobilized cibacron blue F3GA in column chromatography.

Bovine lactoferrin, isolated from colostral milk, interacted strongly with immobilized Cibacron blue F3GA column. Lactoferrin, adsorbed on the dye column, could not be eluted by 8 M urea, 1% Triton X-100, and 75% ethylene glycol, but was eluted by .1 M sodium hydroxide, 1 M potassium thiocyanate, 3 M potassium chloride, and free Cibacron blue F3GA. Electrostatic forces between the sulfonic groups of Cibacron blue F3GA and the basic side-chain groups in lactoferrin molecule probably are responsible for the observed interaction. The elution profile for lactoferrin differed from those of lactoperoxidase and serum albumin, which might allow efficient isolation of lactoferrin from whey via affinity chromatography.