End-group Determination Research Articles

Improved preparation of semisynthetic PheB1-tritiated insulin.

Semisynthetic [PheB1-3H]insulin was prepared. Each intermediate in the synthesis was characterised by HPLC, polyacrylamide gel electrophoresis and end group determination by dansylation. The end product had a specific activity of up to 59 Ci/mmol. Semisynthetic insulin proved indistinguishable from natural insulin in its chemical and immunological properties and was shown to be biologically fully active by in vivo and in vitro assays.

Preparation and properties of porcine relaxin derivatives shortened at the amino terminus of the A chain.

Porcine relaxins shortened at the N terminus of the A chain were produced after protection of all amino groups with the base-labile [[(methylsulfonyl)ethyl]oxy]carbonyl (Msc) protecting group. The first two amino acids were removed by cyanogen bromide digestion whereby simultaneously a free alpha-amino group was generated in position A3. The resulting des-ArgA1,MetA2-N epsilon A7,N epsilon A16,N epsilon B8-tris [[[(methylsulfonyl)ethyl]oxy]carbonyl]relaxin. was further shortened by preparative Edman degradation. The shortest derivative obtained was des-ArgA1,MetA2,ThrA3,LeuA4,SerA5,GluA6 -N epsilon A7,N epsilon A16,N epsilon B8-tris[[[(methylsulfonyl)ethyl]oxy]carbonyl]relaxin. The deprotection of the derivatives in alkaline media resulted in crude des-A(1-2)- to des-A(1-6)-relaxins, which were subsequently purified by gel filtration on Sephadex G-50 superfine followed by either ion exchange chromatography on CM-cellulose at pH 5.1 or high-performance liquid chromatography on reversed-phase columns. During the CNBr digest, a side product was isolated that was identified as the corresponding homoserine ( [HseA2]relaxin) derivative. Shortened relaxin derivatives and [HseA2]relaxin were characterized by reversed-phase chromatography, electrophoresis, end-group determination, and amino acid composition. Circular dichroism studies revealed a distinct change in the structure of relaxins that were shortened by three and more amino acid residues. In the mouse interpubic ligament assay, des-A(1-2)-relaxin and [HseA2]relaxin were fully biologically active while the bioactivity of des-A(1-3)-relaxin dropped to about 50%. Relaxins shortened by four and more amino acid residues were biologically inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex.

Incubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis.

Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein

The in vivo formation of an opioid peptide (exorphin) derived from β-casein has been proved for the first time. It was isolated from duodenal chyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a β-casein fragment by end-group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named β-casomorphin-11, displayed substantial opioid activity in an opiate receptor-binding assay.

Determination of end groups as a function of molecular size for aliphatic polyesters by derivatization of end groups and size-exclusion chromatography with infrared detection

End groups of poly(ethyleneglycol sebacate) having number average molecular weights less than 2500 were characterized as a function of molecular size by derivatizing end groups separately to form 3,5-dinitrobenzoyl and p-nitrobenzyl esters. A hydroxyl end group was reacted with 3,5-dinitrobenzoyl chloride (DNBC) and a carboxyl end group was reacted with O-( p-nitrobenzyl)- N,N′-diisopropyl isourea (PNBD). After separation of these derivatized polyesters by size-exclusion chromatography, the effluent was monitored by using a highly sensitive infrared detector. Concentrations of polyesters were monitored at 1740 cm −1 for a carbonyl group in the main chain, polyesters derivatized with DNBC at 1560 cm −1 for a hydroxyl end group (characteristic absorption band for the nitro group of DNBC), and polyesters derivatized with PNDB at 1537 cm − for a carboxyl end group (a characteristic absorption band for the nitro group of PNBD). By this technique, three types of polyesters having different end groups were characterized: a diol-type polyester, a mixture of polyesters of a diol type and a mono-ol/monocarboxyl type, and a mixture of polyesters of a dicarboxyl type a mono-ol/monocarboxyl type.

Composition of Undigested Peptide Fragments from Gluten with Regard to Bioavailability of Essential Amino Acids

ABSTRACTOur purpose was to isolate and characterize the high molecular weight peptides resulting from the in vitro digestion of wheat gluten in a system simulating actual in vivo conditions, and to determine to what extent essential amino acids were included in these peptides. Large peptides were separated from the smaller products by CuSephadex G‐25, TLE and TLC, treated with dansyl chloride for end group determination, and redansylated to identify the remaining amino acids. Fifteen peptides, hexapeptides or larger, were isolated and were found to contain about 16.1% of the threonine, 15.2% of the methionine and 12.3% of the lysine originally present in wheat gluten.

Separation and characterization of ?-caprolactone oligomers by gel permeation chromatography

It is shown that the combined use of gel permeation chromatography and the isocyanate method of determination of hydroxyl end groups extends the possibilities for studying complex oligomer mixtures of ɛ-caprolactone. Oligomer mixtures of ɛ-caprolactone obtained by (C6H5)3CSbCl6 and by (C6H5)3CK are investigated. At initiation by both the initiators — the cationic and the anionic one — a covalent bond between the initiator and the polymer chain is formed. In the case of the initiation by (C6H5)3CK intramolecular transesterification proceeds which results in cyclic oligomers. At initiation by (C6H5)3CSbCl6 linear oligomers are formed. It is assumed that the ɛ-caprolactone polymerization by (C6H5)3CSbCl6 proceeds by alkyl-oxygen bond scission.

Rapid purification of a phospholipase-free α-bungarotoxin: Maintenance of cation barriers of acetylcholine receptor membranes upon preincubation with purified toxin

The purification of highly homogeneous, phospholipase-free α-bungarotoxin (α-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of α-Bgt is described. The method combines a conventional procedure for the purification of α-Bgt [ D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 243–262 ] with high-resolution gel-filtration and cation-exchange chromatography steps to remove membrane-damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of 125I-α-Bgt. Apparent homogeneity of the purified α-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A 2 activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A 2 activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of 125I-α-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. α-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of α-Bgt. Availability of the new α-Bgt preparation allows for an assessment of the inertness of α-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes.

Peptides of human erythrocyte band 3 protein produced by extracellular papain cleavage.

The structure of the human red blood cell membrane band 3 protein has been investigated by proteolysis of intact cells with papain. Papain cleaves the 35,000-dalton chymotryptic peptide (CH35) of band 3 into two integral fragments of Mr 7,500 (P7) and 28,000 (P28). The papain cleavage of CH35 causes inhibition of band 3-catalyzed Cl transport. The peptide P28 carries the band 3 carbohydrate and also contains the 2 cysteine residues in CH35. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) reacts covalently with a lysine residue on P28, rather than P7, in native band 3. However, if the cells are first digested with papain and then reacted with H2DIDS, there is significant covalent reaction with P7, producing a covalent cross-link between P7 and the 60,000-dalton chymotryptic peptide (CH60). Graded papain digestion experiments and end group determinations indicate that the alignment of the band 3 peptides is N-CH60-P7-P28-COOH. The NH2-terminal sequence of P7 is identical with a segment of a peptic fragment of band 3 recently sequenced (Brock, C.J., Tanner, M.J.A., and Kempf, C. (1983) Biochem. J. 213, 577-586). This published sequence, plus our sequence data on CH35 and P7, show that papain cleaves the outer surface of CH35 at two sites, which are 6 residues and 71 residues from the chymotrypsin cleavage site. The 65-residue peptide (P7) between these sites is the best characterized segment of band 3 thus far described: its sequence and location in the band 3 primary structure are now known, and both ends of the peptide are unambiguously exofacial.

Isolation and amino-acid sequence determination of monkey insulin and proinsulin.

Insulin has been isolated and purified from rhesus monkey pancreas by means of acid-ethanol extraction, gel filtration and ion exchange chromatography. The complete amino-acid sequence of the hormone has been determined by amino-acid analysis of the oxidized A- and B-chains, by end group determination, by the identification of the C-terminal residues (AsnA21 and ThrB30) by carboxypeptidase A digestion and by Edman degradation of the S-carboxymethylated A- and B-chains. The 51-residue monkey insulin was shown to be identical to human insulin. From the known insulin and C-peptide sequence the primary sequence of monkey proinsulin has been proposed.

Amino-acid sequence and disulfide linkages of the anaphylatoxin, des-Arg omega-C5a, from porcine serum.

The primary structure of the porcine complement-derived peptide, des-Arg omega-C5a, has been analysed. Des-Arg omega-C5a is the natural secondary product of the activation fragment of the fifth component of complement, C5a, and represents a classical anaphylatoxin. The elaborated amino-acid sequence confirms the structure of porcine C5a proposed earlier by Gerard and Hugli, with one exception. Further, by end-group determination and sequencing of the unreduced core of des-Arg omega-C5a the position of its three disulfide bridges has been determined, now allowing insight into the tertiary structure of des-Arg omega-C5a.

Determination of vinylester end groups in polyethylene terephthalate by coulometric bromination

A chemical method is described for the quantitative determination of vinylester end groups (VE groups) in polyethylene terephthalate (PETP). The method is based on coulometric bromination of a solution of PETP in di-chloroacetic acid containing potassium bromide, water and mercury(II) chloride. As some impurities present in PETP are also brominated, a correction for this effect is carried out. The standard deviation of the method is 0.25 mmol/kg in the region of 1–20 mmol/kg.

End group determination in hydroxyl-telechelic polyisobutylenes by infrared spectroscopy

A convenient method for the quantitative determination of terminal hydroxy functionality of linear α,ω-di (hydroxy) —polyisobutylene and tri-arm star α,ω,δ-tri(hydroxy) polyisobutylene has been developed and evaluated. The method involves infrared quantitation of the free OH absorption at 3640 cm−1 by the use of long path liquid cells in very dilute CCl4, solution (i.e., in a range where H bridges are absent) in conjunction with Mn determination (vapor phase osmometry or gel permeation chromatography). The results obtained by this method confirm earlier end-group analysis data obtained by more cumbersome methods and indicate that the number of OH end-groups in α,ω-di(hydroxy) polyisobutylene and α,ω,δ-tri (hydroxy)polyisobutylene produced by the inifer technique is 2.0±0.1 and 3.0±0.15, respectively.

Adrenocorticotropin: ACTH1-38 is a major product of biotransformation by brain synaptic membranes.

Since adrenocorticotropic hormone is found in the brain, and several of its fragments affect adaptive behavior, the formation of fragments of ACTH1-39 by a rat brain synaptic membrane fraction was investigated. Following the incubations at physiological pH conditions, the digests were fractionated by HPLC to quantitate the amounts of ACTH1-39 remaining and products formed. Time- and enzyme-dependent disappearance of ACTH1-39 was accompanied by the accumulation of a major peptide metabolite (product B). Amino acid analysis and NH2-terminal end-group determination revealed that product B was identical to ACTH1-38. These results indicate the predominance of carboxypeptidase activity in the degradation of ACTH1-39 by brain synaptic membranes.

Determination of unsaturated end groups in low molecular weight poly(vinyl chloride) by 1H NMR spectroscopy of the hydroxyphenyl substituted product

AbstractPoly(vinyl chloride) of low molecular weight (the fraction extracted with a hexane/acetone mixture (vol.‐ratio 3:2)) was substituted by hydroxyphenyl groups and then analyzed by 1H NMR spectroscopy. It was established that the 1H NMR spectra of hydroxyphenyl substituted poly(vinyl chloride) prepared by removal of HCl, allows to determine the number of unsaturated end groups and to distinguish between the structures CHClCH2CHCHCH2Cl and CH2CHClCHCHCH2Cl, and even between their trans and cis forms. In addition the 1H NMR spectra of hydroxyphenyl substituted poly(vinyl chloride) prepared without removal of HCl allow to determine the total number of unsaturated end groups.

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.

Synthesis and properties of human gamma-lipotropin.

The synthesis of human gamma-lipotropin by the solid-phase method is described. The synthetic product was characterized by Rf in partition chromatography on Sephadex G-50, paper electrophoresis, polyacrylamide gel electrophoresis, thin-layer chromatography, HPLC, end group determination, peptide mapping of a tryptic digest, and amino acid analyses of acid and enzymic digests. The synthetic material is identical to natural human gamma-lipotropin when assayed against natural human beta-lipotropin for lipolytic activity.

Preparation and properties of covalently linked insulin dimers.

The synthesis of six isomeric insulin dimers, linked through selected amino groups of the monomers by a dicarboxylic acid, is described. Symmetrical dimers were obtained by direct crosslinking of N,N-bis(methylsulfonyl-ethoxycarbonyl)insulins with the bis(p-nitrophenyl) esters of dicarboxylic acids. The synthesis of asymmetrical dimers was achieved by use of Msc-protected insulin active ester intermediates. N epsilon-B29,N epsilon B29'-Insulin dimers containing oxalyl, suberoyl and dodecanedioyl crosslinks were produced. N alpha B1,N epsilon B29'-Insulin dimers were suberoyl and dodecanedioyl crosslinks were synthesized; all other dimers were synthesized with suberoyl crosslinks. The positions of crosslinks were determined by sulfitolysis, tryptic digestion, electrophoresis and quantitative end-group determination. The dimers showed potencies between 1-60% that of insulin on a weight basis in stimulating lipogenesis in isolated fat cells. The potencies are considerably lower than the relative binding affinities determined with isolated fat cells.

63] Purification of human fibroblast interferon by high-performance liquid chromatography

This chapter presents a procedure combining the techniques of affinity chromatography and high-performance liquid chromatography (HPLC) for the preparation of fibroblast interferon from serum-containing medium. The purified fibroblast interferon is homogeneous by several criteria of molecular characterization and has a specific activity of approximately 4 x l0 8 units/mg. The purified protein is present in a completely volatile, detergent-free solvent. From several possible approaches, the combination of Blue Dextran-Sepharose affinity chromatography with reverse-phase HPLC at acidic pH is outlined. In contrast to leukocyte interferon, it is not possible to use neutral pH conditions for the HPLC steps because fibroblast interferon rapidly lost activity when organic solvents (propanols, acetone, Methylcellosolve, and such) are present. The data for the homogeneous protein is a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which coincided with the recoverable antiviral activity, and a single protein peak in the last chromatographic step, which coincided with the antiviral activity. The purified preparations are characterized by amino acid analyses, amino sugar analyses, end-group determinations, tryptic maps, and amino acid sequencing.

Various applications of functional group analysis

The importance of functional group analysis is demonstrated with a number of examples in various fields. Special attention is given to the determination of end groups in several kinds of high and low relative molecular mass materials.