A high-throughput method for the quantification of iron saturation in lactoferrin preparations (original) (raw)
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Bactericidal Activity of Human Lactoferrin: Differentiation from the Stasis of Iron Deprivation
Infection and Immunity, 1982
Previous studies have demonstrated a direct iron-irreversible inhibition of a variety of microorganisms by human apolactoferrin. The present study compared the bactericidal effects of lactoferrin on Streptococcus mutans with the bacteriostatic effects of iron deprivation. Growth (as determined by change in optical density) and macromolecular synthesis, as determined by incorporation of 14 C-labeled uracil, thymidine, and lysine, were inhibited by incubation of washed exponential-phase S. mutans NCTC 10449 with purified human apolactoferrin. Similarly, apolactoferrin inhibited glucose uptake and metabolism. Iron-saturated lactoferrin had no effect on bacterial growth or metabolism and was capable of serving as a source of iron in iron-depleted medium. S. mutans failed to grow, and there was no indication of macromolecular synthesis in iron-depleted partially defined medium; however, glucose metabolism continued, though at a reduced rate, and viability was retained for 72 h. There was...
Evaluation of different methods for determination of the iron saturation level in bovine lactoferrin
Food Chemistry, 2014
This work evaluated the most commonly used methods for determining maximum heterotrophic specific growth rates (Pm) in batch reactors. Parallel batch experiments Were conducted under various initial substrate-to-biomass (SoWXo) ratios and values of the solids retention time (0). The maximum specific growth rate, pm, was determined simultaneously according to measurements of oxygen consumption (i.e., oxygen uptake rate, OUR) and volatile suspended solids (VSS) increase. The SOdXO mtio was found to significantly influence pm values. Under high SO/Xo ratios (= 20), fast-growing bacteria seemed to gain a competitive advantage resulting in higher Pm values than those obtained under low Se/XO ratios (= 1.5). The OUR-based estimate of Pm (Pm(OUR)), under certain circumstances, is differentiated from Pm that is based on exponential bacterial growth (Pm(vss)), and seems to be more a measure of substrate oxidation than a measure of bacterial growth. At high SDOXO ratio and low 0 c, 1Im(OUR) was significantly higher than the Pm(vSs)s indicating that considerable uncoupling between anabolism and catabolism was occurring under these conditions. Batch experiments conducted at high Se/Xo ratios seemed to be a more sensitive method for determining Pm values in the presence of an inhibitor than tests conducted at low SOIXO ratios. Ilater Environ. Res., 75, 549 (2003).
Iron-binding fragments from the N-terminal and C-terminal regions of human lactoferrin
Biochemical Journal, 1978
Digestion of lactoferrin with pepsin at pH3.0 gave an iron-binding half-molecule that represents the C-terminal part of the native protein. Tryptic or chymotryptic digestion of 30%-iron-saturated lactoferrin yielded the N- and C-terminal half molecules, which could be separated by DEAE-Sephadex chromatography. The N- and C-terminal fragments did not show any immunological cross-reaction. The carbohydrate of lactoferrin was distributed equally between the two fragments.
BioMetals, 2016
Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.
Lactoferrin and Iron Absorption in Newborn Infants
Pediatric Research, 1987
ABS'I'H.J~('I'. Hesolts frorn csperirncnts in this laborator! using "' l;c suggest that I)o\irle lactoferrin (1.12 has no effect on iron a1)sorption in rats. !t str~d! was thercforc carrieti out in new horn infants to measure the effects of 1.f on iron retention. 13otinc 1,f was I:~l)cled with the stable isotope in I' c and fetl to 7-da>-oltl inktnts in ;I standard milk formula. Iron retention \\;IS estirn;ttetl b) measuring the un-;rl)sorbcd "1;e excreted in the feces during the follol~ing 3 days using neutron ;rcti\ation an;rl\sis. 'l'hc rewits tterc cornp:rrccl with those oht;iined from a group of infants fed ;I similar le\cl of iron a s ferric chloride, 1;lbeled \\ith ' "l.' e. together with 30 mg ;iscorl)ic acid. 'l'hcre was a her! wide v;lriation in percent iron retention amongst the infants but no ovcr:~ll tliffcrcnce bcttr.ccn tlie I,f and ferric chloride groups. 'l'tiis confirms t l~c pre~iorrs findings in rat\ that 1.f does not inflr~cncc thc ;r\ailal)ilit! of nonhcmc iron. (P cclicrtr Kes 22: 651-654, 1087) 1,f, 1;lctoferrin /\AS, atomic ;rt)sorption spcctrornetr! Nit,{, neutron a c t i~a t i o n ;lnalysis
Varying iron release from transferrin and lactoferrin proteins. A laboratory experiment
Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 2017
Iron metabolism is an important subject of study for undergraduate students of chemistry and biochemistry. Relevant laboratory exercises are scarce in the literature but would be very helpful in assisting students grasp key concepts. The experiment described here deals with different iron release mechanisms of two protagonists in iron metabolism: serum transferrin (Tf) and lactoferrin (Lf). Despite having very similar structures and iron-binding sites, Tf releases practically all its iron at pH 5.5 while Lf requires a significantly lower pH of 3. This difference in behavior is directly related to their respective biological functions as Tf blood-borne iron into the cell, while Lf competes with pathogens to sequester iron in biological fluids at more acidic pHs. During this experiment, the students will carry out iron loading and unloading on both human Lf and Tf and monitor the iron release at different pHs using UV-Vis spectroscopy. With this simple approach, the students will dis...
Absorption of lactoferrin-iron in suckling pigs
Nutrition Research, 1983
Lactoferrin is the major iron-binding protein in human milk, and is believed to be of importance in the mechanism of iron absorption. The absorption and retention of iron from iron-saturated bovine lactoferrin was studied in suckling pigs using Fe, and compared to absorption and retention of FeSO 4. Lactoferrin bound iron was absorbed and incorporated into red blood cells to the same extent that sulphate iron. Net iron retention, measured by whole body counting of radioactivity one week after administration of a tracer dose, also showed a similar uptake of lactoferrin bound iron and sulphate iron. The results demonstrate that the iron-deficient suckling pig utilizes iron from lactoferrin at least as well as from an inorganic salt. Uptake data give rise to the hypothesis that the mechanisms for transfer of iron from mucosa to serosa and the incorporation of iron into red blood cells, may differ for the two compounds.
Thermal Denaturation of Human Lactoferrin and Its Effect on the Ability To Bind Iron
Journal of Agricultural and Food Chemistry, 1998
Thermal stability of human milk lactoferrin was studied by differential scanning calorimetry and compared with that of recombinant human lactoferrin produced in Aspergillus awamori. Maximum peak temperature, transition enthalpy, and activation energy of lactoferrin as isolated from human milk (67.0°C, 2276 kJ/mol, and 275.5 kJ/mol, respectively) increased significantly when lactoferrin was fully saturated with iron (90.6°C, 3209 kJ/mol, and 387.6 kJ/mol, respectively) which indicates that the binding of iron to lactoferrin is an important factor in the stabilization of its structure. Similar results were obtained for recombinant human lactoferrin, indicating a high degree of resemblance between both proteins. The ability of human lactoferrin to bind iron after heat treatment was also studied, remaining practically intact after treatments of 72°C for 20 s or 135°C for 8 s, while more severe treatments reduced markedly this ability.