Oligophosphopeptides of varied structural complexity derived from the egg phosphoprotein, phosvitin

Phosvitins are the principal phosphoproteins in the eggs of oviparous vertebrates. They have an exceptionally high serine content and most, or even all, of the serine residues are esterified to phosphate. The phosphorylated residues tend to occur in uninterrupted runs of as many as 28 phosphoserines (as inXenopus phosvitin). This unique structural feature gives phosvitins extraordinary properties and can be expected to play a key role in phosvitin function. For example, the concentration of phosphate groups provides for numerous highly efficient metal-binding sites in clusters. The mode of binding had been shown to be affected by the size of the protein and the degree to which serine residues are phosphorylated. For structure-function studies of phosvitins (and other polyphospho-proteins), phosphopeptides of differentiated structural complexity are desirable. Such model peptides were produced in this work by limited proteolysis of chicken phosvitin, and oligophosphopeptides of widely varying sizes, phosphoserine content, and sequence were purified and characterized. These include phosvitin segments containing one, two, or several oligophosphoserine runs, corresponding to segments of the N-terminal, C-terminal, and core sequence of the protein.     

EXAFS studies of Fe(III)-phosvitin at high metal to protein ratios

The stereochemistry of the Fe(III) binding sites in chicken egg phosvitin (PST) at very high iron content, in solution and as a powder, has been investigated through EXAFS spectroscopy. We found that the EXAFS spectra obtained for aqueous PST solutions at metal:protein ratios of 20:1 and 40:1 are very similar to those previously obtained by us on a Fe₁₀PST sample. In all cases the iron ions are octahedrally coordinated by oxygen atoms of the serine-bound phosphate groups and by other ligands from either the protein or the solvent. The average metal-donor atom distance is 1.94 Å. At variance, the EXAFS results for a Fe₅₀PST powder sample suggest the occurrence of a switch in iron coordination from octahedral to lower coordination numbers (5,4). The average iron-oxygen distance is virtually unchanged; apparently, four iron ligands are provided by four different coordinate phosphate groups from the phosphorylated serine residues abundant in the protein. This finding contains interesting implications for the structure-function relationships of this intriguing protein.     

Phosvitin isolation from fish eggs: methodological improvements including 'specific' phosvitin precipitation with ferric ion

Modifications of the method of Wallace et al. [Can. J. Biochem. 44, 1647-1655 (1966)] for phosvitin isolation from vertebrate eggs were devised to enhance the method's general effectiveness. Phosvitins which do not precipitate on dilution from solutions of their lipovitellin complexes may be selectively adsorbed onto, and desorbed from, DEAE-cellulose. Phosvitins which are too small for dialysis or ultrafiltration may be concentrated or desalted by precipitation with stoichiometric amounts of ferric ions, followed by iron removal with EDTA on gel filtration. Since phosvitin distribution among ammonium sulfate fractions depends on initial protein and salt concentrations in a species-specific manner, pilot experiments are needed to establish conditions for optimal fractionation.     

Iron binding by phosvitins: variable mechanism of iron release by phosvitins of diverse species characterized by different degrees of phosphorylation

The rate of reductive iron release from Fe(III) complexes of phosvitins of diverse fish species, at varied initial degrees of saturation with iron, was studied with particular attention to the effect of the degree of phosvitin phosphorylation on the kinetics of iron release. The reaction was followed colorimetrically as phosphorprotein-bound iron was transferred to an excess of o-phenanthroline, in the presence of hydroquinone as a reducing agent. The principal finding was the variability of the kinetic order or iron release by phosvitins, depending on their degree of saturation with iron and the extent to which their serine residues were phosphorylated. Highly phosphorylated proteins, especially at high initial degrees of iron saturation, obey first-order kinetics. Partially phosphorylated proteins, especially at low initial degrees of iron saturation, release their iron in a zero-order fashion. First-order rates imply that the iron binding sites are kinetically independent of each other. Zero-order behavior appears to reflect iron release from hypothetical iron-binding clusters serving as kinetically effective reactive centers of unchanging concentration for most of the time course of the reaction. Variations of the initial degree of iron saturation of given phosvitins produced variations in their kinetic behavior. The results are considered in terms of a dynamic model of phosvitin iron binding sites which may constitute themselves diversely, in response to the amount of iron that is to be accommodated, or may reconstitute themselves as their molecular environment becomes altered.     

Phosphoprotein phosphatase activity of human prostate acid phosphatase

Human prostate acid phosphatase (EC 3.1.3.2) has been shown to dephosphorylate different phosphoproteins with the maximum rate at pH 4.0-4.5. The activity with phosvitin is distinctly higher than with beta-casein, casein and most of all than with riboflavin-binding protein. The native phosvitin is homogeneous on isoelectric focusing with pI value of 2.1, whereas phosvitin partially dephosphorylated (in about 15%) by the prostate acid phosphatase shows multiple bands with pI values of 3.5 - 6.8 or higher. The phosphate groups bound to serine residues are removed enzymatically twice as fast as phosphothreonine residues. The apparent Km value for phosvitin was 2.4 X 10(-7) M, and is by three orders of magnitude lower than Km of p-nitrophenyl phosphate (2.9 X 10(-4) M). The competitive inhibitors of prostate acid phosphatase, fluoride and L(+)-tartrate, show the same Ki values for phosvitin and p-nitrophenyl phosphate.     

Iron oxidation and transferrin formation by phosvitin.

The catalytic activity of phosvitin in Fe(II) oxidation and the addition of iron to transferrin were studied under various conditions. It was concluded that the Fe(II) oxidized by phosvitin would bind to apotransferrin, although an appreciable fraction of Fe(III) remained bound to phosvitin. Fe(III) also migrated from phosvitin to apotransferrin. This reaction was first-order with respect to Fe(III)-phosvitin concentration with a half-time (t1/2) of 10 min, and a first-order rate constant, k=0.069min-1, in 700 muM-phosphate buffer, pH 7.2, at 30 degrees C. The catalysis of the oxidation of Fe(III) by phosvitin was proportional to O2 concentration, and is quite different from the relative O2 independence of Fe(II) oxidation as catalysed by ferroxidase. A scheme for the mobilization and transfer of iron in the chicken, including the role of ferroxidase, phosyitin and transferrin, is presented.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Iron(III)--phosphoprotein chelates: stoichiometric equilibrium constant for interation of iron(III) and phosphorylserine residues of phosvitin and casein.

Estimates of the strength of iron binding to model phosphoproteins were obtained from equilibrium dialysis experiments. Iron-free phosvitin (chicken and frog) or alpha sl-casein (cow) was dialyzed against the iron(III) chelates of nitrilotriacetate (NTA), )ethylenedinitrilo)tetraacetate (EDTA), or citrate. Protein-bound metal was measured at equilibrium; competition of chelator and phosphoprotein for iron(III) was determined by reference to comprehensive equilibrium equations presented in the Appendix. Analysis of the iron-binding data for phosvitin suggested that clusters of di-O-phosphorylserine residues (SerP.SerP) were the most probable iron-binding sites. A stoichiometric equilibrium constant of 10(18.0) was calculated for the formation of the Fe3+(SerP.SerP) chelate. When comared on the basis of phosphate content, casein bound iron more weakly than phosvitin. However, if the stoichiometric equilibrium constant for the formation of the casein Fe3+(SerP.SerP) chelate (10(17.5) was adjusted to account for the fact that a smaller percentage of casein phosphoserines occurs in di-O-phosphorylserine clusters, the affinity of casein and phosvitin for iron was very similar. A theoretical comparison showed that the "strengths" of the ferric chelates can be ranked: EDTA greater than phosphoprotein di-O-phosphorylserine greater than citrate greater than NTA.     

Iron:phosphorus ratio in surface sediment as an indicator of phosphate release from aerobic sediments in shallow lakes

Analysis of Danish lakes showed that both mean winter and mean summer concentrations of lake water total phosphorus in the trophogenic zone correlated negatively with the total iron to total phosphorus ratio (Fe:P) in surface sediments. No correlation was found between the water total phosphorus concentration and either the sediment phosphorus concentration alone or with sediment calcium concentration. The increase in total phosphorus from winter to summer, which is partly a function of net internal P-loading, was lowest in lakes with high Fe:P ratios in the surface sediment.A study of aerobic sediments from fifteen lakes, selected as representative of Danish lakes with respect to the sediment Fe and phosphorus content, showed that the release of soluble reactive phosphorus was negatively correlated with the surface sediment Fe:P ratio. Analysis of phosphate adsorption properties of surface sediment from 12 lakes revealed that the capability of aerobic sediments to buffer phosphate concentration correlated with the Fe:P ratio while the maximum adsorption capacity correlated with total iron. Thus, the Fe:P ratio may provide a measure of free sorption sites for orthophosphate ions on iron hydroxyoxide surfaces.The results indicate that provided the Fe:P ratio is above 15 (by weight) it may be possible to control internal P-loading by keeping the surface sediment oxidized. Since the Fe:P ratio is easy to measure, it may be a useful tool in the management of shallow lakes.     

Potentiometric and ³¹P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and 31P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP(6) ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P-O-Fe-O-P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)-InsP(6) aggregates. (31)P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T(1) relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP(6) molecule results in an unstable heterogeneous system.     

The effect of various iron hydroxide concentrations on the anaerobic fermentation of sulfate-containing model sewage

The addition of iron hydroxide and iron-reducing bacteria into a fermenter for anaerobic processing of sulfate-containing sewage was shown to decrease sulfate reduction and sulfide concentration, while increasing the total organic carbon (TOC) and methane production. The effect of iron (III) in sulfate-containing sewage depended on its dose, which can be expressed as molar ratio Fe(III)/SO4(2-). Sulfide concentration increased monotonically, reaching 91 mg/l and 45 mg/l after 15 days of processing at Fe(III)/SO4(2-) ratios of 0.06 and 0.5, respectively. However, soluble sulfide production was not observed at ratios equaling 1 and 2. At ratios of 0.06, 0.5, 1, and 2, the maximum rates of TOC removal were 0.75, 1.15, 1.39, and 1.55 g TOC/g of organic matter (OM) per 1 h. Methane production rates were 0.039, 0.047, 0.064, and 0.069 mg/g OM per 1 h, with the mean relative amounts of methane in the biogas being equal to 25, 41, 55, and 62%, respectively. These data can be applied to the development of new methods of anaerobic purification of sulfate-containing sewage.     

Comparison of iron,manganese, and phosphorus retention in freshwater littoral sediment with growth of Littorella uniflora and benthic microalgae

Sediment columns from an oligotrophic lake were percolatedwith artificial porewater in two 46-day experiments toexamine the effects of Littorella uniflora and benthicmicroalgae on retention of phosphorus (P) by either iron(Fe) or manganese (Mn). Cumulative retention of P, Fe, andMn was 2–5 times higher in sediment with L. uniflora thanin sediment with microalgae, because of higher P uptake andmore efficient Fe and Mn oxidation by L. uniflora than bymicroalgae. Thus 34% and 21%of added P was retained in L. uniflora inhabited sediments asmetal-oxide bound P compared to 11% and2% in microalgae inhabited sediments, inexperiments supplied with Fe and Mn, respectively. Theatomic ratio of Fe/P precipitation was about 1 and forMn/P precipitation it was about 5. These ratios indicateprecipitation of Fe(III)-phosphate (strengite) and metastableMn(IV)-compounds containing phosphate and hydroxide ions invariable amounts. In addition to metal-oxide P precipitation,increased P retention in the vegetated sediment was also causedby the presence of humic acid compounds, which accountedfor about 26% of total retained P.     

Potential role of in vitro iron bioavailability studies in combatting iron deficiency: a study of the effects of phosvitin on iron mobilization from pinto beans

Iron deficiency and iron overload affect one billion people worldwide. Treatment of iron malnutrition can be enhanced by an understanding of iron bioavailability from the diet. We have focused on the development of in vitro methods for determining iron bioavailability in the hopes of providing both an understanding of the chemical basis leading to the inhibition or enhancement of iron absorption and the provision of methodologies which will allow nutritionists around the world to ascertain iron bioavailability of local foods and food combinations. The study reported here focuses on the effects of phosvitin, a suspected inhibitor of iron absorption found in egg yolks, on the chemistry of iron during the in vitro enzymatic digestion of pinto beans. Three basic types of information were obtained. First, the total soluble iron was determined during in vitro enzymatic digestion under simulated oral, gastric (pH 2) and duodenal (pH 6) conditions. Phosvitin was found to have a strong solubilizing effect at pH 6 and pH 2 when in the presence of ascorbate. Pyrophosphate also leads to high iron mobilization. A second approach is to determine the static Fe2+ and Fe3+ concentrations following in vitro enzymatic digestion of pinto beans at pH 2 and pH 6. Ascorbic acid enhanced the total soluble iron at both pH values, however, only at pH 2 was a large proportion of the iron found in the Fe2+ state and then only in the presence of phosvitin but not pyrophosphate. A third approach is to determine the amount of Fe2+ formed in the digestive supernatant during a 10-min incubation with ferrozine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the Fenton Reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical (^?OH) formation from H₂O₂ in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited ^?OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the ^?OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H₂O₂. These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.

Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.     

The primary structure of avian phosvitins. Contributions through the Edman degradation of methylmercaptovitins prepared from the constituent phosphoproteins

Methylmercaptovitins were prepared from the constituent phosvitin phosphoproteins as well as from the CNBr cleavage peptides derived from the methionine-containing phosphoproteins. Edman degradation of these methylmercaptovitins has afforded partial sequence information and identifies the serine phosphorylation sites in the phosphoprotein. Primary structure, determined for approximately seventy-seven residues from the amino terminus of the hen methionine-containing phosphoprotein, agrees fully with that deduced from recently published nucleotide sequence data, and provides corrections to results of earlier work on enzymatically dephosphorylated samples. Partial sequence data, together with corrections to earlier results, are also provided for phosphoproteins from duck and turkey phosvitins, as well as for the methionine-free phosphoprotein from hen phosvitin. All phosphoproteins have N-terminal leader sequences of low serine content. Sequences of the methionine-containing group are homologous, as are the sequences of the methionine-free group, but the groups differ significantly from one another. Unphosphorylated sites appear to have a fractional distribution over all available serine residues.     

Phosphorylation of phosvitin by casein kinase-2 provides the evidence that phosphoserines can replace carboxylic amino acids as specificity determinants

The consensus sequence of casein kinase-2 consists of a serine (threonine) followed by a cluster of glutamic and/or aspartic acids, the one at position +3 playing an especially crucial role (Marin et al., (1986) Eur. J. Biochem. 160, 239-244 and Kuenzel et al. (1987) J. Biol. Chem. 262, 9136-9140). None of the 123 serines of the main phosvitin component (34 kDa) fulfils such a requirement (Byrne et al. (1984) Biochemistry 23, 4275-4279), rather, most of them are clustered into stretches of up to 14 entirely phosphorylated residues. Three out of the four threonines lie close to the N-terminal side of such phosphoseryl blocks. Here we show that native 34 kDa phosvitin is a poor substrate of casein kinase-2, its radiolabeling occurring mostly at threonine residue(s); a very slight (1%) previous dephosphorylation with acid phosphatase converts phosvitin into an excellent substrate for casein kinase-2, its phosphorylation occurring almost exclusively at serine residues. Extensive dephosphorylation however (greater than 40%) reduces the phosphorylation efficiency of casein kinase-2. These results show that phosphoserine residues can replace carboxylic residues as specificity determinants for casein kinase-2.     

The effect of various iron hydroxide concentrations on the anaerobic fermentation of sulfate-containing model wastewater

The addition of iron hydroxide and iron-reducing bacteria into a reactor for anaerobic processing of sulfate-containing wastewater was shown to decrease sulfate reduction and sulfide concentration, while increasing the total organic carbon (TOC) and methane production. The effect of iron (III) in sulfate-containing wastewater depended on its dose, which can be expressed as molar ratio Fe(III)/SO ₄ ^2? . Sulfide concentration increased monotonically, reaching 91 and 45 mg/l after 15 days of processing at Fe(III)/SO ₄ ^2? ratios of 0.06 and 0.5, respectively. However, soluble sulfide production was not observed at ratios equaling 1 and 2. At ratios of 0.06, 0.5, 1, and 2, the maximum rates of TOC removal were 0.75, 1.15, 1.39, and 1.55 g TOC/g of organic matter (OM) per 1 h. Methane production rates were 0.039, 0.047, 0.064, and 0.069 ml/g OM per 1 h, with the mean relative amounts of methane in the biogas being equal to 25, 41, 55, and 62%, respectively. These data can be applied to the development of new methods of anaerobic purification of sulfate-containing wastewater.     

Inhibition of zinc absorption by iron depends on their ratio

Previous studies upon zinc-iron interactions gave conflicting results that could come from differences in protocol design or in trace element status of subjects. The present work assessed the influence of zinc : iron ratio and iron deficiency upon zinc absorption. The digestive absorption of zinc sulphate (100 mol Zn/l) in presence of iron gluconate was studied in perfused jejunal loops (n = 6/group) of normal rats (range 0–1000 mol Fe/l) and iron deficient rats (200–750 mol Fe/l). In normal rats no significant iron inhibition on zinc absorption occurred at Fe:Zn ratio below 2:1. At higher ratios zinc uptake and net absorption decreased significantly (p<0.05). Between 2:1 and 5:1 a dose dependent inhibition of zinc absorption occurred and reached a plateau beyond this ratio. In iron deficient animals no changes in zinc uptake, mucosal retention and absorption compared to normal animals occurred at ratio 2:1. At higher ratios differences were observed at every zinc absorption step except for mucosal retention at 7.5:1 ratio.

Iron-zinc interactions depend on their ratio and on previous trace elements status of subjects. Due to the wide and unknown variations that were likely to occur between the subjects of previous human and experimental studies, these results could explain some of the discrepancies between their results.