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Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 degrees C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta-lactoglobulin hydrolysis were assessed by using various beta-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.

Multiple forms of mature alpha(s1)-casein have been characterized in ovine variants A and D using a combination of mass spectrometry and automated Edman degradation. Mature ovine alpha(s1)-casein was found to be a heterogeneous mixture of at least seven molecular species. The main component, representing about 50% total alpha(s1)-casein, corresponded to the full-length (199 residues long) protein. The other components were alpha(s1)-casein of different lengths: 198 (less Gln78), 191 (less peptide 110-117), 191 residues (less peptide 140-148), 190 (less peptide 110-117 and Gln78), 190 (less peptide 140-148 and Gln78), and 183 (less peptides 110-117 and 140-148) residues long alpha(s1)-casein. Each of the alpha(s1)-casein multiple forms occurred at three different phosphorylation levels, due to the partial phosphorylation of both Ser115 (at about 50%) and Ser41 (at about 20%). In the case of deleted peptide 110-117, the protein heterogeneity linked to the partially phosphorylated Ser115 was abolished, and only two levels of phosphorylation were observed. These multiple forms differing in molecular weight and degree of phosphorylation may have been developed from an exon skipping during mRNA splicing in ovine alpha(s1)-casein, similar to that recently described in the case of its caprine counterpart.

The primary structure of syringicin (syr), a new acidic α-elicitin, isolated from culture filtrates of Phytophthora syringae, causal agent of citrus fruit rot, has been determined using a combined approach 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 Mr 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. © 2001 Elsevier Science Ltd. All rights reserved.

The reaction products of epichlorohydrin with human α- and β- 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 α-globin was much more reactive than the β-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 α-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. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations: ECH epichlorohydrin Hb hemoglobin GC/MS gas chromatography/mass spectrometry ESMS electrospray mass spectrometry MALDI-MS matrix-assisted laser desorption/ionization mass spectrometry MS/MS tandem mass spectrometry LC liquid chromatography RP-HPLC reversed-phase high-performance liquid chromatography TFA trifluoroacetic acid SIM selected ion monitoring u, mass unit. The standard one-or three-letter code has been used for the amino acids.

The 13-globin gene cluster of the domestic sheep (Ovis aries) shows two common haplotypes: the A haplotype, which bears the adult sA allele (HBBA), and the B haplotype, which bears the adult sB allele (HBBB) [1, 2]. The chromosomal organization of the A haplotype was found to be similar to that present in goat (Capra hircus) since it shows the same triplication (5’ Er_Ea_NJsI sc_E[u_Erv_Jsrr_sn_£v_Evr_ NJs,rr-sF-3’) of an ancestral four gene set (E-E-NJs-s) [3]. This set is characterized by the presence of two embryonic genes (e), one pseudogene (NJs), and one adult gene (s). The expression of the adult gene varies during ontogenic development and under different physiological conditions. The sc, sA, and sF genes are, in fact, expressed during juvenile, adult and fetal life, respectively, and the sc gene expression is reactivable, at the expense of sA gene, under particular physiological or experimental conditions such as anemia and hypoxia or the administration of erythropoietin [4-6]. The B haplotype is considered to have diverged from the A haplotype, as the result of a recent deletion from a triplicated locus. In fact, due to the lack of the whole juvenile four-gene set containing the sc gene, it is duplicated (5’ er srr NJsr sB Errr-EIV yrsrI sF-3’) and sheep which are homozygous for the sB allele do not exhibit the sBia>scswitching

Elicitin 172, an acid protein with elicitor activity, has been isolated in true form from culture filtrates of Phytophthora nicotianae , the causal agent of crown and root rot of tomato ( Lycopersicon esculentum ). The M r (10,349±1) of the purified protein, determined by ES–MS, is identical to that calculated for parasiticein using the mean isotopic composition and assuming the occurrence of three disulfide bridges. The primary structure of elicitin 172, determined using also MALDI–MS experiments, shows complete identity with parasiticein, with elicitin 310 and a cloned elicitin gene from P. parasitica (= P. nicotianae ), confirming conservation of the elicitin sequence within a single species. The protein induces necrosis (hypersensitive reaction) on tobacco, but no symptoms on tomato, when applied on the leaves. Tomato pretreated with elicitin 172 was affected by P. nicotianae , as well as by the phytotoxic aggregates, naturally occurring with the elicitin in the non permeated dialysis fraction of culture filtrates. Finally, the elicitin induce protection of capsicum ( Capsicum annuum) and vegetable marrow ( Cucurbita pepo ) from P. capsici .

The present study demonstrates that NO produced in vitro by inducible nitric oxide synthase in red cells can convert hemoglobin contained in the red cells to S-nitrosohemoglobin. Experiments carried out either in the absence or in the presence of a low molecular weight thiol, such as cysteine, showed that in the first case the target of NO is heme-Fe2+. On the contrary, in the presence of cysteine, the first step is the formation of S-nitrosocysteine, followed by transfer of the NO group to a particular cysteine residue of β-globin, cysteine 93. These results confirm previous data indicating the preferential formation of S-nitrosohemoglobin at that site by chemical methods [Ferranti et al. (1997) FEBS Lett. 400, 17–24], and the existence of a physiological mechanism of inactivation for NO circulating in blood. The analysis of S-nitrosohemoglobin can also allow the quantification of the NO levels in blood to be applied for in vitro and in vivo measurements.

A procedure for the determination of haemoglobin adducts formed by exposure to methyl bromide was evaluated as a possible method for the measurement of a biological index of exposure to the alkylating agent. The reaction products after in vitro incubation were used to design the chemical syntheses of deuterated peptides corresponding to the tryptic peptides where the modified residues had been identified. These peptides were used as standards for the quantitative evaluation of real samples. The correlation coefficient was r = 0.998 in the range 2.5-20 ppm. The relative standard deviation was about 3%. Blood samples were digested with trypsin and the mixture was analysed by liquid chromatography/electrospray mass spectrometry through selected ion monitoring of the mass signal relative to the modified peptides. The analysis of blood samples from workers exposed to methyl bromide demonstrated the usefulness of this mass spectrometric-based method for the monitoring of human exposure to the genotoxic alkylating agent via the synthesis of suitable peptide standards. This procedure is the first alternative method to the well established monitoring of N-terminal adducts, the latter not being applicable to all alkylating agents.

The primary structure of purified SV-IV, a major secretory protein synthesized by the rat seminal vesicle (SV) epithelium, was analysed by electrospray mass spectrometry (ES-MS). The protein was found to be highly heterogeneous. The various components were separated and identified by reversed phase high-performance liquid chromatography (HPLC) on line with ES-MS. Structural characterization of the SV-IV cyanogen bromide digests revealed the occurrence of a Val/Met substitution in about 50% of the purified protein molecules. We suggest that this mutation is the expression of a genetic polymorphism. Other minor components, corresponding to structural changes (fragmentation, deletion, and phosphorylation) of SV-IV and probably due to post-translational modifications of the native protein, were also detected. In particular, by using protein tyrosine phosphatase hydrolysis combined with ES-MS, we demonstrated that, in the phosphorylated species of SV-IV, a single phosphate group was covalently bound to the Tyr-36 residue. The significance of these findings in relation to the regulation of important biological processes, such as immune response, blood coagulation, inflammatory reaction, and mammalian reproduction, are discussed. © 1997 John Wiley & Sons, Ltd.

La matiere azotee soluble dans le tampon citrate 0,2 mol/L a pH 8,0 de deux echantillons de 14 et 38 mois de Grana Padano, un fromage italien a pâte dure, a ete diafiltree sur membranes a porosite de 3 et 10 kDa. Deux fractions ont ete recueillies, contenant des peptides de masse moleculaire inferieure a 3 kDa et superieure a 10 kDa. La fraction inferieure a 3 kDa a ete fractionnee par chromatographie liquide haute performance (CLHP) en phase inverse sur colonne C18 et les peptides identifies en combinant la spectrometrie de masse a bombardement rapide et la degradation d'Edman. La plupart des 91 peptides identifies dans cette fraction A du fromage de 14 mois provenaient des caseines α s1 (44 peptides) et β (32 peptides). La majorite de ces derniers presentaient une origine commune dans la region 1-105/107 de la caseine β. Trente peptides resultaient de la degradation du peptide α s1 -CN (fl-79) forme par action de la plasmine sur la caseine α s1 . Treize peptides provenaient de la caseine α s2 et un seul de la para-K-caseine. Trente-huit peptides ont ete identifies dans la fraction inferieure a 3 kDa du fromage de 38 mois (dont 21 peptides identifies dans le fromage jeune): 8 issus de la caseine β, 27 de la caseine α s1 , 1 de la caseine α s2 et 2 de la para-κ-CN. La fraction superieure a 10 kDa a ete soumise a un fractionnement sur colonne Mono Q, puis a une separation par CLHP en phase inverse sur colonne C4. Les peptides ont ete identifies en combinant la spectrometrie de masse a l'electrospray et la degradation d'Edman. Les resultats concernant une fraction chromatographique sont presentes: les variants B et C de la caseine α s1 et plusieurs fragments de grosse taille tels que le peptide α s1 -CN-I ont ete identifies. Les resultats montrent que les differentes techniques de spectrometrie de masse permettent de caracteriser, mieux que d'autres techniques,la nature des peptides issus des caseines, quelle que soit leur taille.

Recent studies have demonstrated the biological importance of the interaction of S-nitrosothiols, which can be considered as nitric oxide (NO) protein donors, especially haemoglobin, at the level of Cys residues. It was recently proposed that S-nitrosohaemoglobin is formed within red blood cells and serves as a regulatory function. In human haemoglobin the alpha-subunit contains one Cys residue and the beta-subunit contains two Cys residues, one of which (beta-Cys93) is highly reactive and conserved among species, although its function has remained unknown. Electrospray ionization mass spectrometry was used to monitor the results of exposure of haemolysates to S-nitrosocysteine under different conditions and thus addressed some aspects of NO-haemoglobin interaction. When an equimolar ratio of S-nitrosothiol was added to haemoglobin, only a single NO molecule was added. Peptide mapping by liquid chromatography-mass spectrometry located the nitrosyl group at the level of beta-Cys93 demonstrating that this was the preferred site of formation of S-nitrosohaemoglobin. The present data also suggest that electrospray mass spectrometry can allow quantification and characterisation of S-nitrosoproteins in blood.

The identity of multiple forms of caprine alpha(s1)-casein in variants A, B, and C has been determined by structural characterisation using mass spectrometry, automated Edman degradation and peptide mapping. Mature goat alpha(s1)-casein exists as a mixture of at least four molecular species which differ in peptide chain length. The main component corresponds to the 199-residues form already described. The other three, in lesser amounts, were shorter forms of alpha(s1)-casein and differed for the deleted peptides 141-148, as shown previously for ovine alpha(s1)-casein, peptide 110-117, or Gln78. Analysis of alpha(s1)-casein mRNA from milk somatic cells demonstrated that these forms originated from skipping events at the level of exon 13 (codifying for peptide 110-117) and 16 (codifying for peptide 141-148) and from the presence of a cryptic splice site within exon 11 (whose first CAG triplet encodes Gln78) during primary transcript processing. The finding of these splicing abnormalities in the three common variants A, B, and C suggests that this is a general feature of alpha(s1)-casein in goat. A further source of heterogeneity of caprine alpha(s1)-casein was identified in the discrete phosphorylation of seryl residues. Eight serine residues (at positions 44, 46, 64 to 68 and 75) are fully phosphorylated (except in variant A because of the replacement Glu77-->Gln which prevents phosphorylation of Ser75). Conversely, Ser115 and Ser41 are phosphorylated only to about 50% and 20%, respectively. Ser12, although located in a consensus triplet, is never phosphorylated, similarly to the ovine alpha(s1)-casein variants. These results confirm that there are stabilised mechanisms of simultaneous synthesis of alpha(s1)-casein at different length and of post-translational modification in both caprine and ovine species.

Electrospray ionization mass spectrometry has been used to study the possible non-covalent interaction between oligonucleotides and beauvericin (B) mycotoxin. Beauvericin-oligonucleotide adduct formation was observed even at low mycotoxin concentration (25 pmol/microL). Adducts were found with different numbers of B ligands attached. The selectivity of binding of B ligands to two different oligonucleotides has been shown to be similar indicating that beauvericin does not have a strongly preferred base sequence or base site in the DNA. In a competitive complexation reaction, beauvericin forms specific adducts with oligonucleotide while another mycotoxin, nigeromicin, which causes apoptosis without fragmentation of DNA, does not.

Casein phosphopeptides (CPP) which develop in Grana Padano cheese at different ages were isolated by precipitation with Ba2+ and analysed by HPLC. Profiles were complex throughout the period between 4 and 38 months. CPP in a cheese sample 14 months old were identified by a combination of fast atom bombardment–mass spectrometry and Edman degradation. They were found to consist of a mixture of components derived from three parent peptides, β-CNf(7–28)4P, αs1-CNf(61–79)4P and αs1-CNf(7–21)4P. In total, 45 phosphopeptides were identified: 24 from β-CN, 16 from αs1-CN and 5 from αs2-CN. The presence of aminopeptidase activity during cheese ripening was deduced from the presence of a number of CPP of different lengths with the loss of one or more residues from the N-terminus. The longest had C-terminal lysine and seemed to be progressively hydrolysed by carboxypeptidases A and B to shorter peptides. CPP in cheese appeared to be shortened plasmin-mediated products. Moreover, those most resistant to further hydrolysis contained at least three closely located phosphoserine residues. The anticariogenic activity of CPP is also discussed.

The microheterogeneity of the paracelsin mixture broth of Trichoderma reesei was analysed using mass spectrometric methods, in particular high energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Based on the liquid secondary ion mass spectrum of the mixture, there are three main components, with molecular masses M and (M +/- 14), together with two minor components of molecular weight (M +/- 28). The high-energy CID tandem mass spectra of both the protonated and sodiated molecules yielded abundant and characteristic fragment ions, but of very different types. It was found that a paracelsin peptaibol in a mixture could be successfully sequenced based on the tandem mass spectra of its protonated and sodiated molecules or, alternatively, on the tandem mass spectra of its y(7) and b(13) fragment ions. A terminology for indicating these sequential peptide fragments is proposed. To determine the sequence of new analogues, tandem mass spectra of the y(7), (y(7) +/- 14), b(13), (b(13) +/- 14) and (MH +/- 14) positive ions were also taken. Based on these experiments, four new paracelsin components (PA-F, PA-G, PA-H and PA-I) were sequenced successfully. The microheterogeneity of the mixture was found to be more pronounced than had been assumed previously. In these new analogues, besides positions 6 and 9, position 17 is also involved in the exchange. MS/MS studies on minor fragment ions, such as (b(13) - 28) and (b(8) - 14) show further microheterogeneity at positions 3, 5, 10 and 12, which increase the number of possible minor components. (C) 1997 by John Wiley & Sons, Ltd.

A mass spectrometric procedure is described for the structural study of the adducts formed in human hemoglobin by in vitro exposure of erythrocytes to the alkylating agent methyl bromide using different protein to reagent ratios, Peptide mapping by HPLC and tandem mass spectrometry allowed location of methylated amino acids within the protein sequence, A prominent reactivity of several nucleophilic side chains in human hemoglobin subunits was observed, which was modulated by the concentration of the alkylating agent, Cysteine residues, the main reactive sites, were fully methylated in hemoglobin exposed to a 10-fold excess of methyl bromide, differently from other residues, including histidines, showing a heterogeneous pattern of methylation that was largely directed by their environment, No evidence of methylation was found at the heme proximal histidines beta 92 and alpha 87. A more selective methylation was obtained when the ratio methyl bromide: hemoglobin was lowered to about 1:1, In this last case only specific residues were reactive, Among them, the N-terminal amino group of both alpha- and beta-globins, cysteine 104 in the alpha-chain and cysteine 93 (not cysteine 112) in the beta-chain, indicating a different accessibility to reaction of the sulfhydryl groups on the protein chain, Thus hemoglobin side chains are selectively modified and the degree of modification at each site is a function of the position of the single amino acid residue within the protein quaternary structure, raising the possibility that alterations of structure and functional properties of human hemoglobin following exposure to alkylating agents may be mediated through such covalent protein modifications, The results obtained demonstrate the usefulness of the analytical approach for the characterization of hemoglobin adducts with methyl bromide or similar compounds, which can constitute the basis for biomonitoring of human exposure.

An analytical procedure is described for the identification of the adducts formed by interaction of methyl bromide and haemoglobin. The reaction products of in vitro incubation of haemoglobin with methyl bromide have been characterised by electrospray mass spectrometry and gas chromatography-mass spectrometry. A prominent reactivity of several potential nucleophilic sites of haemoglobin was observed. Analogous results were recorded on blood samples of workers exposed to methyl bromide. The results obtained represent the basis for the complete structural characterisation of the modified haemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of alkylation degree and the identification of modified amino acids in proteins.

Publisher Summary The combination of capillary zone electrophoresis (CZE) with fast atom bombardment mass spectrometry (FAB/MS) analysis according to the FAB mapping procedure couples a potentially fast high-resolution separation method with one of the most powerful techniques for structural characterization. Capillary electrophoresis has the ability to handle very small volume samples (picoliter range) and attomole (10 -18 mol)-level detection limits reaching separation efficiencies of the order of 10 6 theoretical plates. The CZE of proteins with UV detection have often resulted in broad, tailing peaks because of the absorption of proteins on active sites of the negatively charged capillary wall. However, the introduction of coated capillaries made CZE a fast and sensitive method for the detection of even similarly charged variants; the resolution obtained using this technique is comparable to the well-established electrophoretic and chromatographic procedures and requires smaller samples. Its speed and simplicity with respect to the given techniques and the possibility of automation and of development on a micropreparative scale mean the whole procedure is a significant alternative to conventional systems.

1. The amino acid substitution which characterizes the haemoglobin I variant from sheep has been ascertained using a combination of Fast Atom Bombardment mass spectrometry and protein sequencing. 2. A Ser for Gly substitution at position 13 (10 of the A helix) was found in a polypeptide with the overall sequence of the beta B globin. 3. On the basis of the nucleotide sequence of the beta B-globin gene, a C to T transition occurring on a CpG doublet is considered to be responsible for the amino acid substitution. 4. This represents the first observation of a variant sheep Hb due to a mutation which is rather common in the human genome. 5. Amongst ruminants, serine is normally present at position 13 of goat and sheep epsilon II and gamma chains and of bovine gamma chain which had an independent and more ancient evolutionary origin than the beta chains.

The present study describes how mass spectrometry was extensively applied to the characterization and quantification of modified amino acids within the polypeptide chain of Angiotensin I, chosen as model substrate, combining the use of fast atom bombardment mass spectrometry with gas chromatography--mass spectrometry. The reaction products after in vitro incubation of Angiotensin I with styrene oxide, a well known carcinogen, under different conditions, have been characterized: a prominent reactivity of several potential nucleophilic sites of Angiotensin I was shown, including two histidine residues and a tyrosine residue; it is worth noting that it has never been stated that tyrosine is highly reactive with styrene oxide. The results obtained demonstrate the usefulness of mass spectrometry for the structural determination of chemically modified amino acids in peptides and proteins, and the presence of a reliable relationship between reaction conditions and the production of alkylated amino acids. This characterization procedure offers the possibility of identifying reactive sites following exposure to unknown alkylating agents.

Hb City of Hope [beta 69(E13)Gly----Ser] was detected by reversed phase high performance liquid chromatography in an asymptomatic carrier from Naples, Southern Italy. The amino acid substitution, identified by fast atom bombardment mass spectrometry, was due to a TGG----TGA substitution as assessed by DNA sequencing. Analysis of the chromosomal background indicates that the globin gene cluster containing the mutant gene has most probably been rearranged by a recombination event, since the mutation was associated with restriction fragment length polymorphism haplotype IX, instead of haplotype I, as previously reported.

This paper describes a simple and rapid analytical method for the structural identification of abnormal human hemoglobins. Globin chains obtained by precipitation of erythrocyte hemolysate in cold acetone are directly analyzed by capillary zone electrophoresis in coated capillaries without any prior treatment. The speed and the high resolving power of capillary zone electrophoresis allow fast differentiation of hemoglobins with similar charges. Capillary zone electrophoretic tryptic mapping has also been performed for each globin, so that complete variant characterization can be achieved by direct comparison of the variant tryptic map with the corresponding normal one. Coupling electrophoretic data with analysis of enzymatic digests by mass spectrometry according to the “fast atom bombardment mapping” procedure makes it possible to quickly identify amino acid variations. This paper describes how the method can be applied to the characterization of common and uncommon variants and underlines the advantages and limitations of the procedure along with its potential uses in structural analysis of proteins.

Hb. Heathrow [beta 103(G5)Phe- greater than Leu] was identified in an Englishman with a life-long history of polycythemia, his father had been similarly affected. A hemoglobin variant was suspected from the high oxygen affinity of the patient's blood. The Hb Heathrow abnormal beta chain was resolved from the normal beta chain by high performance liquid chromatography, and the abnormal peptide and the amino acid replacement were identified by mass spectrometry. The corresponding base change (C- greater than G at codon 103) was demonstrated by sequence analysis of the polymerase chain reaction amplified exon 2 of the genomic beta-globin gene. This is only the third known instance of Hb Heathrow.

A rapid mass spectrometry procedure, based on direct mixture analysis using conventional electron ionization in both the low and high resolution modes, was used to match pigments present on a counterfeit $100 United States bank note with pigments contained in two inks suspected of having been used. The results demonstrated that the pigments present on the false bank note and those contained in the inks were the same.

1. 1. Hemoglobins from three phenotypes of Italian water buffalo ( Bubalus bubalis ), named AA, AB and BB, were selected by starch gel electrophoresis at alkaline pH and analyzed using polyacrylamide gel isoelectric focusing and subsequent analysis of titration curves to reveal differences between two types of hemoglobin identified as Hb fast and Hb slow. 2. 2. Globins from Hb fast and Hb slow were purified by fast protein liquid chromatography (FPLC). Electrophoretic differences were found in the respective α-chains using polyacrylamide gel disc-electrophoresis at acid pH, polyacrylamide gel isoelectric focusing and by subsequently analyzing titration curves. 3. 3. The results suggest that the α chains of Hb fast and Hb slow, called II α and II α , respectively, differ in at least two aminoacid residues. Subsequently, these amino acids were identified as lysine and cysteine.

We have characterized a beta 112 Arg hemoglobin in an individual from Naples, Italy, with minimal clinical problems. Blood tests revealed only slight reticulocytosis and hemoglobin instability. Furthermore, high value of alkali resistance tests for Hb F were observed. Isoelectricfocusing of globins showed the occurrence of a band migrating between the normal alpha and beta globin chains. The fairly stable variant chain was purified by fast protein liquid chromatography. A mass map of the tryptic digest was obtained by fast atom bombardment mass spectrometry clearly showing that we were dealing with a beta chain variant. However, the peptide 105-120 was missing and two new ones were present, i.e.: 105-112 and 113-120; we assumed these peptides to be generated because of the substitution of 112 Cys with an arginine residue. Further confirmation stemmed from the fast atom bombardment mass spectra of the tryptic digest submitted to a single Edman degradation step and to carboxypeptidase B further hydrolysis. The beta-globin chain variant was thus mass mapped to an extent of about 98%. Such a variant, named Hb Indianapolis, was first reported by Adams et al, as an extremely unstable variant producing the phenotype of a severe beta-thalassemia. Contrary to the findings of the above authors the occurrence of the same variant in a clinically normal individual from a Spanish family has recently been reported. Because the clinical manifestations in the latter case are similar to those observed by us, the conclusion can be drawn that beta 112 Arg hemoglobin is not a biologically unstable variant but should be regarded as belonging to the class of unstable hemoglobins giving rise to only marginal clinical problems.

The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-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.

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. γ2- and γ3-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. Copyright © 2000 John Wiley & Sons, Ltd.

Occurrence of free phosphoserine (SerP) in ripened cheeses was investigated to clarify whether this amino acid can be directly released from casein by enzymatic attack. Free amino acids extracted from cheese were separated by IEC or RP-HPLC and the peak of SerP always eluted close to unretained material and acidic components. Presence of these interferences suggests that the content of free SerP (from 0.8 to 16.4 mmoles/kg cheese) could be highly overestimated. These figures were dramatically lowered (up to 100 times) after a purification step of the cheese extract on cationic column, but the chromatographic separation of SerP was not yet interference-free. However, these values of free SerP are not significant on the basis of the total amount of free amino acids (

A centripetal temperature gradient takes place in Grana Padano (GP) during moulding because of the slow heat transfer within the cheese and the fast cooling of the outer part. This gradient, in combination with the low pH value, induces a centripetal inactivation of alkaline phosphatase (ALP). Throughout the ripening process, the activity of the enzyme keeps > 3 10(5) mU/kg cheese in the peripheral zones and

Sustainable sources of dietary proteins are making their way into the market to reduce the impact of food production on the environment. Considerations from several standpoints are to be made when supporting the dietary shift toward these alternative sources, particularly regarding their nutritional and safety quality. Research is making enormous steps forwards by producing highly valuable proteins with little environmental impact or by turning by-products and wastes into sources of valuable proteins. The chapter will summarize the environmental, nutritional, and safety aspects related to novel foods, including microalgae, plants, insects, microbial, and synthetic-based ingredients. Consumer acceptability is taken into central consideration throughout the chapter being the final user of the novel food ingredients.

Global food security is threatened by reduction of agricultural resources and by an increasing world-wide population. Given this future context, traits relating to yield stability and sustainability are the major focus of plant breeding. Nowadays, plant breeding represents a key component involved in global food security. The process of plant breeding is theoretically simple, but its power resides in the fact that it creates novelty where to select. Crop productivity is a quantitative trait and, as such, bases on a complex genetic mechanism and on a strong environment influence. From the simple plant sexual crossing, the way to enhance crop productivity has been subject to profound innovations giving birth to the molecular plant breeding, a discipline that integrates molecular biology, biotechnology and genomic research and support plant breeders to enhance complex traits. Here, we describe all the emerging genetic technologies that can be used to study and exploit genetic diversity in relation to crop productivity enhancement. In particular, we gave a general view on the genetic basing crop productivity, the traditional methods used for its study and the reasons that lead researchers to develop newer technologies.