Degradation of ß-casomorphin-7 through in vitro gastrointestinal and jejunal brush border membrane digestion.

This work aimed to study the opioid peptide ß-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

Grape skin phenolics as inhibitors of mammalian α-glucosidase and α-amylase--effect of food matrix and processing on efficacy.

Type-2 diabetes is continuously increasing worldwide. Hence, there is a need to develop functional foods that efficiently alleviate damage due to hyperglycaemia complications while meeting the criteria for a sustainable food processing technology. Inhibition of mammalian α-amylase and α-glucosidase was studied for white grape skin samples recovered from wineries and found to be higher than that of the drug acarbose. In white grape skins, quercetin and kaempferol derivatives, analysed by UPLC-DAD-MS, and the oligomeric series of catechin/epicatechin units and their gallic acid ester derivatives up to nonamers, analysed by MALDI-TOF-MS were identified. White grape skin was then used for enrichment of a tomato puree (3%) and a flat bread (10%). White grape skin phenolics were found in the extract obtained from the enriched foods, except for the higher mass proanthocyanidin oligomers, mainly due to their binding to the matrix and to a lesser extent to heat degradation. Proanthocyanidin solubility was lower in bread, most probably due to formation of binary proanthocyanin/protein complexes, than in tomato puree where possible formation of ternary proanthocyanidin/protein/pectin complexes can enhance solubility. Enzyme inhibition by the enriched foods was significantly higher than for unfortified foods. Hence, this in vitro approach provided a platform to study potential dietary agents to alleviate hyperglycaemia damage and suggested that grape skin phenolics could be effective even if the higher mass proanthocyanidins are bound to the food matrix.

Characterization of heat-labile toxin-subunit B from Escherichia coli by liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation.

Short communication: molecular genetic characterization of ovine alpha(S1)-casein allele H caused by alternative splicing.

Sequencing of ovine CSN1S1*H cDNA showed an absence of exon 8 in comparison with GenBank sequences; the absence was confirmed by protein sequencing. We demonstrated that this allelic aberration is the result of a deletion of 4 nucleotides, the last 3 of exon 8 and the first 1 of intron 8, which are replaced by an insertion of 13 nucleotides in the DNA sequence. The insertion is a precise duplication of a part of the adjacent intronic sequence of CSN1S1*C''. These sequence differences result in an inactivation of the splice donor sequence distal to exon 8, leading to upstream exon skipping during the serial splice reactions of the ovine CSN1S1*H pre-mRNA, and may affect the specific casein expression as well as protein characteristics.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution.

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Formation of structured polymers upon controlled denaturation of beta-lactoglobulin with different chaotropes.

Prolonged exposure (>90 days) of bovine beta-lactoglobulin (BLG) to subdenaturing concentrations of either urea or potassium thiocyanate resulted in the formation of ordered polymers in the form of fibrils. The fibrils obtained with each chaotrope showed major differences in morphology, surface properties, thiol accessibility, and stability to dissociating agents as a consequence of the different chemical bonds involved in their stabilization. Hydrophobic interactions between BLG monomers are predominant in thiocyanate-formed fibrils, whereas urea-formed fibrils are stabilized by intermolecular disulfides generated through a thiol-disulfide exchange reaction. The different features of fibrils obtained with each chaotrope relate to the peculiar structural features and chemical properties of the "active" monomers generated by subdenaturing chaotrope concentrations in the early phases of the polymerization process, as detected by spectroscopic and limited proteolysis/mass spectrometry studies in the earliest stages of the action of individual chaotropes. The chaotrope-specific features of these early intermediates in turn affect the polymerization mechanism, whose intermediates were studied by size-exclusion chromatography on the soluble fraction at different times of fibril formation. The potential of these findings for the production of protein-derived nanostructures having different and controlled geometries and chemical properties is also discussed.

Caseinomacropeptide self-association is dependent on whether the peptide is free or restricted in kappa-casein.

There is a general agreement that the experimentally determined molecular weight (MW) of caseinomacropeptide (CMP) is greater than the theoretical MW. Some studies suggest that this is due to a pH-dependent aggregation of monomeric CMP. How this aggregation is influenced by pH is not understood. This study was carried out to study the nature of CMP aggregates and to clarify which conditions affect aggregation of CMP. The apparent MW of CMP at different pH values was determined using size-exclusion chromatography. Caseinomacropeptide was further characterized by immunochemical analysis, sodium dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilino-naphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of kappa-casein (CN), and 2 commercial CMP products. Both commercial products and CMP resulting from chymosin-hydrolysis of kappa-CN (at pH 6.6) had elution volumes with a MW corresponding to 35 kDA at pH 8.0 and 3.4. Caseinomacropeptide prepared from pepsin-hydrolysis of kappa-CN (at pH 2.5) eluted as multiple peaks with apparent MW of 35, 18, and 9 kDa, again independently of pH. Hydrolysis of kappa-CN with chymosin or pepsin at different pH values (pH 2.5, 3.4, and 6.6) produced differently sized aggregates of CMP, largely depending on the pH of the hydrolysis. These results indicate that, whereas CMP molecules are irreversibly associated, CMP in kappa-CN may associate reversibly in a pH-dependent manner. We suggest that interactions between para-kappa-CN parts of the kappa-CN molecules may be a requisite for the pH-dependent dissociation/association.

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis.

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins.

Syringicin, a new alpha-elicitin from an isolate of Phytophthora syringae, pathogenic to citrus fruit.

The primary structure of syringicin (syr), a new acidic alpha-elicitin, isolated from culture filtrates of Phytophthora syringae, causal agent of citrus fruit rot, has been determined using a combined approch based on Edman degradation and MALDI-MS (TTCTT TQQTA AYVAL VSILS DSSFN QCATD SGYSM LTATA LPTTA QYKLM CASTA CKTMI TKIVS LNAPD CELTV PTSGL VLNVY SYANG FSSTC ASL). Syr has 98 amino acids with a M(r) of 10194.6+/-0.2, which was determined by electrospray ionisation-mass spectrometry (ES-MS) and in agreement with three disulphide bridges, located between Cys3-Cys71, Cys27-Cys56 and Cys51-Cys95. Syr induces a hypersensitive response and electrolyte leakage in tobacco. These are characteristic elicitor properties of the group and in agreement with the molecular mechanism recently proposed for this kind of protein. Finally, its possible applications in biological agriculture and biomedicine are briefly discussed.

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins.

Primary structure analysis of the four river buffalo alpha-globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two alpha-globin chains, Ialpha1 and IIalpha3, which differ at positions 129 and 131: Ialpha1 has 64 Ala, 129 Phe, 131 Asn; IIalpha3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two alpha-globin chains, Ialpha2 and IIalpha4, which differ at positions 10 and 11: Ialpha2 has 10 I1e, 11 Gln, 64 Asn; IIalpha4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic alpha-globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo alpha-globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.

Phosphorylation of seminal vesicle protein IV on Ser58 enhances its peroxidase-stimulating activity.

In this study we show that SV-IV, a major immunomodulatory, anti-inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES-MS) analysis demonstrated that approximately 10% of the native SV-IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV-IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium-dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV-IV on Ser58 did not produce any effect on the anti-inflammatory properties of SV-IV, as measured by its ability to inhibit the phospholipase A2.

Mass spectrometric characterisation of proteins in rennet and in chymosin-based milk-clotting preparations.

The protein composition of natural rennet and of chromatographic and crystalline chymosin preparations has been defined by on-line reverse-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS) and by tandem mass spectrometry (MS/MS). Natural rennet was found to consist of six chymosin species, corresponding to chymosin A and B genetic variants, each of which comprised a mixture of two other forms differing at theN-terminal end, with one being three residues longer, and the other two residues shorter, than the mature chymosin. Two main tissue proteins were also identified as lysozyme (isozyme 2 plus a novel isozyme labelled 4) and bovine serum albumin. In addition to the proteins, chymosin fragments 247-323 and 288-323 were consistently present in natural rennet. Conversely, chromatographic and crystalline chymosin preparations lacked bovine serum albumin and/or lysozyme, although they contained the same six chymosin species as natural rennet. Since these tissue-specific contaminating proteins each possess specific functions in terms of stabilising enzyme solutions and protecting proteins from proteolytic enzymes, oxidising agents and bacterial proliferation, the rennet may be considered as a functional enzyme preparation that is effectively and naturally adapted to the purposes of cheesemaking. In practice, the highly complex protein composition inherent to natural rennet provided the possibility to differentiate the natural product from other bovine chymosin-based milk-clotting preparations examined in this work.

Similar efficacy following four weeks treatment of asthmatics with formoterol 12 micrograms b.d. delivered by two different dry powder inhalers: differences in inhaler handling.

This randomised, multicentre, parallel-group study compared the clinical efficacy and ease of handling of two dry powder inhalers delivering the long-acting beta 2-agonist formoterol. After run-in, 200 asthmatics on treatment with inhaled corticosteroids and still presenting with suboptimal asthma control were randomised to receive 12 micrograms formoterol twice daily via either the Aerolizer inhaler (Foradil Aerolizer) or the Turbuhaler inhaler (Oxis Turbuhaler) for four weeks. Study variables included the mean morning pre-medication peak expiratory flow (PEF) during the last seven days of treatment and the correct inhaler handling according to inhaler-specific checklists. The mean difference in the effect on morning pre-medication PEF was 13.86 l/min in favour of formoterol via the Aerolizer inhaler (90% confidence interval 2.50, 25.21) in the intent-to-treat population. Eighty-six per cent of the patients under treatment with formoterol via the Turbuhaler inhaler performed correctly all the essential inhalation manoeuvres, whereas 98% of those on the Aerolizer inhaler did so. These results strongly suggest similar clinical efficacy with twice daily treatment of formoterol 12 micrograms metered dose delivered either by the Aerolizer, or the Turbuhaler device. They also suggest that handling the Aerolizer is easier than that of the Turbuhaler.

A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry.

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.

Mass spectrometric identification of a candidate biomarker peptide from the in vitro interaction of epichlorohydrin with red blood cells.

The reaction products of epichlorohydrin with human alpha- and beta- globins, obtained through in vitro incubation of these compounds and red blood cells, were determined by using reversed-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization tandem mass spectrometry. The alpha-globin was much more reactive than the beta-globin. At low incubation ratios, approximating the order of magnitude of epichlorohydrin concentration as found in workplaces, the only modified peptide still detectable was the 62-90 belonging to the alpha-chain and carrying an incremental mass of 92 u on either His72 or His89. Given that the two peptides co-eluted in a single chromatographic peak during RP-HPLC separation, they could be chosen as suitable biomarkers for quantification in the setting up of a new methodology for the biological monitoring of persons occupationally exposed, replacing currently known procedures.

Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation.

The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.

Immunochemical evaluation of bovine beta-casein and its 1-28 phosphopeptide in cheese during ripening.

Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.

Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4.

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of beta-casein (beta-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of beta-CN f7-14, f47-52, and f169-175 and kappa-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the beta-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC(50)s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC(50)s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.

Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry.

Based on analysis by liquid chromatography/electrospray ionisation mass spectrometry, we have developed a new method for fast and sensitive fingerprinting of gliadins and glutenins in wheat flour. Using this procedure the two protein fractions from seven durum wheat varieties have been analysed by high resolution high performance liquid chromatographic separation coupled to accurate determination of molecular mass. In this way, the molecular mass of the single components from both gliadin and glutenin fractions were measured and more than forty components were detected for each fraction indicating a high heterogeneity. Although the chromatographic profiles were similar, the molecular masses of protein components with similar retention times among the varieties were often different. The difference ranged from a few mass units corresponding to single amino acid substitution(s) up to thousands implying peptide deletion or insertion along the protein chain. Two components representing about a half of the gliadin fraction, e.g. gamma(2)- and gamma(3)-gliadin, were identified through the N-terminal sequence and molecular mass determination. We suggest the use of the high level and the molecular mass of these gliadin components as markers to detect traces of wheat in gluten-free food preparations for celiac patients.