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Single-dimension separations are routinely coupled in series to achieve two-dimensional separations, yet little has been done to simultaneously exploit multiple dimensions during separation. In this work, simultaneous chromatography and electrophoresis is introduced and evaluated for its potential to achieve two-dimensional separations. In simultaneous chromatography and electrophoresis, chromatography occurs via capillary action while an orthogonal electric field concurrently promotes electrophoresis in a second dimension. A novel apparatus with a dual solvent reservoir was designed to apply the concurrent electric field. Various compounds were used to characterize the apparatus and technique, i.e., vitamins, amino acids, and dyes. Improved separation is reported with equivalent analysis times in comparison to planar chromatography alone. The feasibility of simultaneously employing chromatography and electrophoresis in two dimensions is discussed. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The analytical potencies of ligand-exchange capillary electrophoresis ( LECE) in regard to the determination of bioactive substances are discussed. As it is shown, complexation with copper(II) in the electrophoretic determination with UV detection makes possible the determination of amino acids, amines, and sugars, which do not absorb in the UV region, with the limits of detection 5-10 mg/L, and reduce the detection limits of the absorbing analytes, such as tryptophane, tyrosine, and histamine by 2-3 times. Prospects for using online preconcentration for the reduction of c are shown on an example of aliphatic amino acids, i.e., thirtyfold preconcentration is attained. LECE is shown to be applicable to the simultaneous determination of glucose, Na(), and K() in human blood serum. Also versions of ligand-exchange capillary electrophoresis with direct UV detection and capillary zone electrophoresis with indirect one are compared in terms of efficiency, time of analysis, and limit of detection. The potencies of the LECE method are compared with those of ligand-exchange chromatography. [ABSTRACT FROM AUTHOR]

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid. [ABSTRACT FROM AUTHOR]

Two techniques for identifying the peaks of phenylthiohydantoin (PTH) amino acids separated under the conditions of micellar electrokinetic chromatography were compared. The first technique is linear regression analysis, in which the retention time of an amino acid is a function of the retention times of two retention-time standards. The second technique takes into account hydrophobicity constants log D′, which were calculated using the ACD/LC Simulator 8.0 program package from ACDLabs (Canada). These constants provide an opportunity to calculate the relative migration times of PTH amino acids taking into account the velocity of the electroosmotic flow. The first technique allows us to identify the electrophoretic peaks of all 16 amino acids separated; the second procedure allows us to predict the elution order of the electrophoretic peaks; the use of a correlation equation gives better results. [ABSTRACT FROM AUTHOR]

Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization-time-of-flight MS and electrospray ionization-quadrupole-time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti-C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis. [ABSTRACT FROM AUTHOR]

An endo-β-1,4-glucanase (EG) was purified from ripe strawberry (Fragaria × ananassa Duch.) fruit using cellulose affinity chromatography. The purified enzyme gave a single protein band of 54 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this protein showed strong homology with the proteins encoded by recently identified EG genes from different strawberry cultivars and from Arabidopsis, pepper and tomato. The enzyme specifically cleaved the β-1,4-glucosyl linkages of xyloglucan but was unable to hydrolyze those of insoluble cellulose. The pH optimum and Km of the enzyme against the artificial substrate carboxymethylcellulose (CMC) were pH 5.0–7.0 and 1.3 mg ml–1, respectively. To assess the role of the Cel1 enzyme in fruit softening a cDNA of the corresponding fruit-specific and ripening-enhanced strawberry gene, cel1, was used to down-regulate cel1 gene expression in transgenic strawberry plants. In several primary transformants, cel1 mRNAwas strongly suppressed in ripe fruit. However, the EG activity and firmness of these fruit were indistinguishable from those of control fruit. The expression of a second gene, cel2, encoding a different strawberry EG was unaltered in the fruits of these transformants. The presence of the cel2 transcript in transgenic plants may have prevented the specific down-regulation of cel1 from revealing its role in fruit softening. [ABSTRACT FROM AUTHOR]

We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1–196 (domain I), 1–404 (domain I+11a) and 1–506 (domain I+IIab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan-(I–404) and the -(1–506)- peptide was successful, but in the case of tile perlecan-(1–196)-peptide no recombinant protein was produced. lmmunoblotting sbowed that both the (l–404)-peptide and (1–506)-peptide are recognized by 95J 10, a monoclonal antibody that was previously raised against perlecan-(24–404)-peptide expressed in Escherichia coli Gel permeation and anion-exchange chromatograhy were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan-(1–404) peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan-(1–404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan (1–404)- peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirus- derived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future. Kewords: perlecan; structure; baculovirus expression; chondroitin sulfate; basic fibroblast-growth factor [ABSTRACT FROM AUTHOR]

A new sulfur-rich basic polypeptide. so called ω-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified ω-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of ω-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteotytic cleavage. ω-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by elcctrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues. and has strong sequence identity (66%) with the sorghum SIα1 α-amylase inhibitor. ω-hordothionin. like γ-hordothionin. exhibited translation inhibitory activity on both cukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to γ-hordothiomn, ω-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. ;γ-hordothionin also inhibited the α-amylase activity from human saliva, while ω-hordothionin and the other different genetic variants of thionins, α- hordothionin and β-hordothionin. failed to show any inhibitory effect. [ABSTRACT FROM AUTHOR]

High-molecular-weight (high-MT) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 151 mg from 8.1 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/ polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelctrophoresis. The molecular weight of horse high-MT kininogcn was estimated to be 78000 by dodecylsullate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-MT, kininogen with bovine and horse plasma kallikreins, several fragments winch contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disullide bonds. The molecular weight and amino acid composition of the heavy chain anti the light chain from horse high-MT kininogen were very similar to those of the heavy and light chains from bovine high-MT kininogen. respectively. From these results, it was revealed that horse high-MT kininogen is quite similar to bovine high-MT kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable. [ABSTRACT FROM AUTHOR]

A protein with properties similar to apoprotein A-IV of rat high-density lipoproteins has been isolated from the lipoprotein fraction (e < 1.006 g/ml) of individual non-fasting hypertriglyceridaemic subjects. The Mr of human apolipoprotein A-IV determined by polyacrylamide-gel electrophoresis in sodium dodecylsulfate and by column chromatography in 6 M guanidine hydrochloride is 46000. The amino acid composition is significantly different from any previously characterised human apolipoprotein but resembles that of rat apolipoprotein A-IV. The N-terminal amino acid of human and rat apolipoprotein A-IV is glutamate or glutamine. The pI of the major human A-IV band in 6 M urea is &ap; 5.15. Immunochemically apolipoprotein A-IV is not identical with any of the well known human apolipoproteins. Human apolipoprotein A-IV is present in all fractions of e < 1.006 g/ml isolated by a single ultracentrifugal spin from non-fasting subjects, but is lost from lipoproteins of e < 1.006 g/ml upon recentrifugation. Irnmunoelectrophoretic studies show that most of human apolipoprotein A-IV is present in the fraction of e < > 1.21 g/ml and is unassociated with the major lipoprotein fractions in serum. Charge- shift electrophoresis however clearly demonstrates the hydrophobic character of human apolipoprotein A-IV. There is evidence that apolipoprotein A-IV is a constituent of chylomicrons that is removed from the particle surface when chylomicrons enter the plasma compartment. [ABSTRACT FROM AUTHOR]

The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63500 and from gel filtration through columns of Sephadex G-100, 58000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 μmol NAD+ incorporated × min-1 × mg-1 of protein at 37 °C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the Km for NAD+ was 23 μM. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

Lysozyme (mucopeptide N-acetylmuramylhydrolase) from Asterias rubens was obtained in a chromatographically and electrophoretically pure state by gel filtration and affinity chromatography. The quantitative amino acid composition, the molecular weight and the N-terminal sequence determined by a sequencer are reported. This new invertebrate enzyme presents important differences when compared to previously studied lysozymes. [ABSTRACT FROM AUTHOR]

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O- [32P]phosphoserine and O-[32p]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major ≈ 66-kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7–3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical ≈ 66-kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE- Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino-terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine-residue sequence spanning the Arg-Gly-Asp cell-binding site of the rat protein osteopontin, was identified in the ≈ 66-kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compo- sitions. Several smaller-molecular-mass phosphoproteins ranging in size over about 55–29 kDa were also observed in the HCl extracts of bone. Peptide mapping of these species demonstrated that the ≈ 66-kDa, ≈ 55-kDa, and ≈ 45-kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the ≈ 66-kDa species since this phosphoprotein remained the prevalent species after a 24-h chase. Because of the predominant association of all the smaller-molecular-mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24-h chase, these results suggested that the lower-molecular-mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the ≈ 66-kDa component in the extracellular matrix. In summary, these results demonstrate that cultured osteoblasts synthesize a major 66-kDa phosphoprotein that is identical to the major HCl-extractable phosphoprotein present in postnatal chick bone. [ABSTRACT FROM AUTHOR]

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pl 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4), These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, Mr could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase. [ABSTRACT FROM AUTHOR]

Alkaline degradation of fusigen, a sideramine from fungi, yields the known monohydroxamic acid fusarinine. L-Ornithine and cis-5-hydroxy-3-methylpentenoic-2-acid occur in fusigen in equal amounts, and ornithine is the only amino acid. Amino group(s) with pK 7,1, but no free carboxyl group can be found in fusigen. The calculated equivalent weight is 280, which again points to fusarinine as a structural subunit. By acetylation of fusigen three products are obtained, which can be separated by electrophoresis or chromatography. The titration data of the partially acetylated fusigen and the molecular weight estimation by vapour pressure osmometry of the desferriform of the fully acetylated fusigen show that fusigen is a trimeric fusarinine. These data together with those obtained from IR- and NMR-absorption spectra lead to the proposed structure: three fusarinine molecules are esterified head-to-tail to build a 36-membered ring. The quantitative estimation of all hydroxamic acids in the culture fluid of Fusarium cubense from the 2nd to the 9th day of fermentation and structural studies on some of these compounds revealed the spontaneous degradation of fusigen by ring opening and sequential split-off of fusarinine units. An analogous degradation pathway starts from N-acetyl-fusigen. The Δ2-anhydromevalonic lactone, found in the cultures of several fungi, seems to stem from the degradation of fusarinine. The total yield of Δ2-anhydromevalonic lactone can be increased severalfold by treatment of the culture fluid with hydrochloric acid prior to extraction. [ABSTRACT FROM AUTHOR]

1. A simple and mild procedure is described for the isolation of equine growth hormone from frozen pituitary glands. it involves water extractions of the glands at pH 7.2 and 9,5, fractionation by ammonium sulfate precipitation and gel filtrations on a Sephadex G-100 column. The yield is 1.7 mg growth hormone per gram of fresh glands with a biological specific activity of 1.6 USP units/mg. 2. The protein obtained is homogeneous judged by disc electrophoresis, ultracentrifugation, gel electrofocusing, gel filtration and end-group analyses 3. A weight-average molecular weight of 20 200 was determined by sedimentation equilibrium analysis. Comparative values were found by gel electrophoresis and exclusion chromatography. Its Stokes radius and frictional ratio were 2.25 nm and 1 21, respectively. 4. The amino acid composition of equine growth hormone shows considerable analogy with those of bovine, ovine and porcine growth hormones. [ABSTRACT FROM AUTHOR]

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further purification of the peptides was carried out by gel filtration on Sephadex G-25 or by high-voltage paper electrophoresis at pH 3.6 and 6.5. Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptie peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio-Gel P-4, and their ammo acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino-terminal region of the inhibitor consisting of AB amino acid residues. The reactive site (Arg63-Ile64) of the inhibitor to trypsin was involved in this region. [ABSTRACT FROM AUTHOR]

A new method is described for the isolation of the subunits of porcine luteinizing hormone which have not been characterized before. By chromatography on SE-Sephadex C-25 in 8 M urea, the two subunits LHα and β were obtained in good yield and without apparent cross contamination. The physicochemical and immunological properties of these subunits were studied. Unlike the bovine or ovine LHβ subunits described earlier, porcine LHβ subunit does not contain lysine. Porcine LHα subunit is electrophoretically heterogeneous and two components represented by two homogeneous electrophoretical zones were isolated and characterized. The amino acid composition of these components is very similar; they differ, however, slightly in peptide maps of their tryptic digests. Whole LHα subunit was submitted to specific cleavage at methionine residues by cyanogen bromide and a homogeneous peptide not containing homoserine was isolated. The latter represents most probably the C-terminal fragment of LHα subunit. Its partial amino-acid sequence and composition is: Gly-Asn-Ala-Arg-Val-Glu-(His3,Lys,CM-Cys3-4,Asp,Thr2,Ser,Glu,Tyr2)-Ser. The fragment contains 34% of sugar (by weight). In view of the similarity in peptide maps and the unambiguous sequence described above, the primary structure of the different components of LHα subunit must be very similar. [ABSTRACT FROM AUTHOR]

The purification of eleven neurotoxins from the venom of three scorpions (Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestreatus) has been performed by Sephadex G-50 filtration (with and without recycling) and equilibrium chromatography on Amberlite CG-50, CM-Sephadex C-50 and DEAE-Sephadex A-50. All the chromatographic steps were carried out in ammonium acetate buffers. Purity of the neurotoxins was assessed by amino acid analysis and zone electrophoresis. All have a molecular weight of about 7000 daltons and do not contain methionine. As shown by amino acid composition and chain length scorpion neurotoxins show a high degree of homology. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

The ℵ-chains were prepared by chromatography and rechromatography of pig S-sulfoimmunoglobulin on Sephadex G-100 and SE-Sephadex, and characterized by starch-gel electrophoresis, amino acid analysis, molecular weight (25 000 ± 2500) and N-terminal amino add (alanine). From the tryptic digest of the ℵ-chains, the N-terminal tetracosapeptide was isolated and its amino acid sequence determined as Multiples equation(s) cannot be represented by ASCII text. These results provide evidence of the similarity between pig immunoglobulin ℵ-chains and the human and mouse type K Bence-Jones proteins. [ABSTRACT FROM AUTHOR]

This paper is the first one of a series devoted to the primary structure of the major protein of cow's milk, αs1-casein, a single-chain phosphoprotein, formerly reposed to have arginine as an NH2-termimal, and Leu-TrpOH as COOH-terminal residues. ryptic digestion of maleyl αs1-casein (genetic variant B), in theory bruited to arginyl bonds, yields eight peptidic fragments, Tm1 to Tm8, which were fractionated by column chromatography and, in some cases, more thoroughly purified by preparative paper electrophoresis and/or chromatography. The ammo acid composition and the phosphate content of these fragments were determined. The fragments Tm3 and Tm8 arise from a partial non specific cleavage of Tm5. Thus, six fragments only—2, 4, 5, 6 and 7—arise from the specific cleavage of the αs1-casein chain at arginyl bonds, a result in accordance with the reported number of six arginyl residues in the chain, one being NH2-terminal. The fragment Tm7, with two arginyl residues, and Tm2 devoid of arginine appear to rcpresent respectively the NH2- and COOH-terminal parts of the αs1-casein chain. These peptidic fragments—except Tm4 which is devoid of lysine—were further digested by trypsin at lysyl bonds, after eliminating the blocking maleyl groups. The resulting peptides were purified by the same methods as mentioned above, and the composition of these tryptic peptides was established. As a control, the tryptic peptides of αs1-casein were simultaneously purified by the same methods from a hydrolysate of the protein which had not been treated with maleic anhydride. In summary, this first step of our work gives: (a) the composition of ail the bovine αs1-casein tryptic peptides; (b) partial information on the location of these peptides in the protein chain: (c) confirmation that the molecular weight of the bovine αs1-casein monomer is approximately 23600. [ABSTRACT FROM AUTHOR]

The article presents abstracts on analytical chemistry including "Separation and quantification of amino acids by capillary zone electrophoresis," "Microfabricated electrochemical detector for chromatography," and "Crystallic silver amalgam for electrochemistry."

Amino acids (AA) are essential nutritional components of a balanced diet and occur in foods in either the free AA form or as the building blocks of proteins. The analysis of AAs in foods is composed of a number of unit operations; the release of the AAs from the food matrix, the separation of the individual AAs and their quantification using calibration standards. Each of these steps has their own idiosyncrasies, e.g. different hydrolysis conditions are required for the optimal release of different AAs and there are a diverse number and type of food matrices, such that most laboratories adapt methods to best suit their applications. There is currently no official standardised method for AA analysis, although the Association of Analytical Communities (AOAC) has validated methods for a number of individual AA components. The established analytical techniques of HPLC (ion exchange or reversed phase) and GC-MS have recently been supplemented by a number of new methods. These include capillary electrophoresis MS and Ultra HPLC-MS, and LC with other detectors. This review will address the intricacies and concerns of the protein hydrolysis step, discuss what specifications or prerequisites need to be placed on the existing and new methods and laboratories using these methods, comment on whether one method can successfully satisfy the exacting requirements of the various unit operations, and finally pose the question ‘Is there any merit in ‘developing’ a validated (e.g. AOAC) official method of analysis for AAs in food?’ [ABSTRACT FROM PUBLISHER]

The article presents news briefs from the pages of Nature, published many in the fiftieth and hundred year edition. In the 50 years ago section, a new and rapid technique of characterizing the chemical properties of a protein is discussed. In the 100 years ago section biometricians are urged to see that the problems they seek to elucidate are sound from a biological point of view. A failure in this regard is highlighted.