bluuebunny/crossref_metadata_2025 huggingface dataset:
ASSOCIATION‐DISSOCIATION AND DENATURATION BEHAVIOUR OF AN OLIGOMERIC SEED PROTEIN α‐GLOBULIN OF SESAMUM INDICUM L. IN ACID AND ALKALINE SOLUTIONS
Authors: Prakash V. , Nandi P. K.
Published: August 2010

The association‐dissociation and denaturation behaviour of the major protein fraction,α‐globulin of sesame seed (Sesamum indicum L.), in acid and alkaline solutions in the ranges of pH 4.2–1.5 and pH 7–12 have been studied. The results of gel filtration, fluorescence and viscosity measurements indicate dissociation and denaturation of the protein up to pH ˜ 3. The difference spectrum in this region arises from a combination of dissociation, denaturation and charge effect on the chromophore. In still stronger acid solution, reassociation of the dissociated fraction takes place by hydrophobic interaction. In alkaline solution dissociation takes place around pH 8, and above pH 10 dissociation and denaturation proceed simultaneously as has been evidenced by sedimentation, fluorescence, spectral change, optical rotation and viscosity measurements. The phenolic group (pKInt = 10.6) in the protein is abnormal and denaturation in alkaline solution is irreversible. Above pH 11.5 further dissociation of the protein takes place. Characteristic pH values of transition from 10.6–10.8 indicate that the transition of the protein involves a single step in alkaline solution.
STUDIES ON THE PARTIAL SYNTHESIS OF PROTEIN ANALOGS BY DIRECT COUPLING TO TERMINAL HOMOSERINE LACTONE DERIVATIVES:
Authors: Dyckes D.F. , Kini Hema , Sheppard R.C.
Published: August 2010

Treatment of basic pancreatic trypsin inhibitor (BPTI, I) with cyanogen bromide smoothly yields the chain cleaved derivative II. The utility of the seco‐lactone (II) in the partial synthesis of protein analogues has been investigated. It is shown that the homoserine lactone ring is sufficiently reactive to combine directly with the radiolabelled synthetic peptide glycylglycylalanine t‐butyl ester in both aqueous and non‐aqueous solution, leading to a BPTI analogue which has been purified and characterized.
THIOLYSIS OF Nim‐2, 4‐DINITROPHENYL‐HISTIDINE PEPTIDES WITH 2‐MERCAPTOETHANOL
Authors: Goren H. Joseph , Fridkin M.
Published: August 2010

The thiolysis of Nim‐2, 4‐dinitrophenyl‐histidine peptides with 2‐mercaptoethanol demonstrates an optimal rate between pH 8.5 and 9.0. Base lability of both reactants and product was investigated as a possible cause for the pH optimum. N‐2, 4‐Dinitrophenylimidazole, a model for Nim‐2, 4‐dinitrophenyl‐histidine peptides, 2‐mercaptoethanol and the product of thiolysis, 2, 4‐dinitrophenyl‐S‐mercaptoethan‐2‐ol, were subjected to aqueous basic conditions (pH 8.5 to 12.0). The reactions were followed spectrophotometrically, and products were identified by comparison of their ultra‐violet spectra with commercially available or synthetic compounds. Mechanisms and rate constants for base hydrolysis of 2, 4‐dinitrophenyl‐thio ethers are presented. Although N‐2, 4‐dinitrophenylimidazole and 2, 4‐dinitrophenyl‐S‐mercaptoethan‐2‐ol dohydrolyze, it is the oxidation of 2‐mercaptoethanol to 2‐mercaptoethanol disulfide which results in the decreasing rate of thiolysis above pH 9.0.
SOLID PHASE SYNTHESIS OF PORCINE LH‐RELEASING HORMONE WITH HYDROGEN CHLORIDE IN FORMIC ACID AS THE DEBLOCKING REAGENT*
Authors: Yonezawa Hiroo , Takahashi Norio , Ohno Motonori , Izumiya Nobuo
Published: August 2010

The decapeptide corresponding to the amino acid sequence of porcine luteinizing hormone‐releasing hormone (LH‐RH) which involves 1 mol of tryptophan was synthesized via solid phase synthesis with two different deblocking procedures which used hydrogen chloride in formic acid and hydrogen chloride in acetic acid containing 1% 2‐mercaptoethanol. After some fundamental studies on the former reagent with respect to deblocking efficiency toward the Boc group, 0.5 M hydrogen chloride (a 10‐fold molar excess with respect to the N‐terminal Boc group) in formic acid was used in the present synthesis. The two synthetic products exhibited the same chemical and biological properties as an authentic LH‐RH. Hydrogen chloride in formic acid has proved effective without a scavenger although loss of peptide from the resin occurred to a somewhat greater extent than that with hydrogen chloride in acetic acid. A derivative of the synthetic LH‐RH formylated at the indole nitrogen had a greatly diminshed biological activity, indicating that the intact indole side chain is essential for the activity.
STRUCTURAL STUDIES ON THE SUCCINYLATED BOVINE SERUM ALBUMIN
Authors: Chang Tiang‐Shing , Sun Siao Fang
Published: August 2010

An analysis of succinylated bovine serum albumin showed that all of the 62 α‐amino groups of lysine residues, 13 hydroxyamino acid residues and 12 tyrosin residues were succinylated, but the succinylated tyrosines were later deacylated. Structural properties of the modified protein have been studied with circular dichroism and sedimentation velocity. At neutral pH (pH 7.60) and in a salt‐free aqueous solution the modified protein is in an expanded form and its helical content is only 30% of that of unmodified protein. The increase of ionic strength restores the original conformation of the protein, whereas the increase of pH further disorganizes the structure of the protein. The results suggest that the electrostatic force alone is responsible for the compact structure of the protein molecule. The same mechanism is believed to underlie the effect of H3O+ and the effect of succinylation of side chain groups on the conformation of bovine serum albumin.
NON‐ELASTOMERIC POLYPEPTIDE MODELS OF ELASTIN
Authors: Rapaka Rao S. , Okamoto Kouji , Urry D. W.
Published: August 2010

The synthesis of two polyhexapeptides is reported. The polyhexapeptides are H‐(Val6‐Ala1‐Pro2‐Gly3‐θ4‐Gly5)n ‐Val‐OMe where θ= Vol or Lys with a mole ratio of 3.5:1 and H‐(Val6‐Ala1‐Pro2‐Gly3‐Lys4‐Gly5)n‐Val‐OMe. The first polymer was utilized with a previously synthesized polyhexapeptide, H‐(Val6‐Ala1 ‐Pro2‐Gly3‐θ‐Gly5)n‐Val‐OMe where θ was either Val4 or Glu4 at a mole ratio of 3.5:1, to obtain an intermolecular cross‐linked matrix by means of primary amide bond formation between the γ‐carboxyls of the Glu residues of one copolymer and the α‐amino groups of the Lys residues of the other copolymer. The cross‐linking reaction was run during a temperature‐elicited phase separation with flow orientation of the polymers. An insoluble, non‐elastomeric, cross‐linked, polyhexapeptide matrix was obtained. The nature of the insoluble polyhexapeptide matrix was well‐demonstrated by the polymer, H‐(Val6‐Ala1‐Pro2‐Gly3‐θ4‐Gly5)n‐Val‐OMe where θ4 is Vol or Lys, which could be formed into cellophane‐like, non‐elastomeric sheets which would tear and which could be shown by microscopy to have sharp edges. This very different property of the polyhexapeptide of tropoelastin as compared to the elastomeric polypentapeptide of tropoelastin is discussed in terms of a different structural role.

The purity of key intermediate hexamers and of the polyhexapeptides is demonstrated by carbon‐13 magnetic resonance in addition to the usual analytical methods.
REMOVAL OF PROTECTED PEPTIDES FROM THE MERRIFIELD RESIN BY POTASSIUM CYANIDE CATALYZED TRANSESTERIFICATION
Authors: Moore Graham , Mcmaster Denis
Published: August 2010

Potassium cyanide catalyzed transesterification with methanol, ethanol or benzyl alcohol was found to be effective for the removal of a variety of protected amino acids and peptides from the Merrifield resin after solid phase peptide synthesis. A peptide was released from the solid support as the methyl, ethyl or benzyl ester by stirring the resin in the appropriate dry alcohol at room temperature for 24 h in the presence of 1% potassium cyanide. No racemization of the C‐terminal amino acid was observed in the transesterification of Z‐Ala‐Val‐resin to Z‐Ala‐Val‐OBzl. The possible occurrence of cyclization and αåβ shift of aspartyl residues was investigated for transesterification reactions in benzyl alcohol. Potassium cyanide catalyzed transesterification with benzyl alcohol has been used for the synthesis of arginine vasotocin. When carried out in 95% aqueous alcohol, the peptide‐resin bond was first transesterified and subsequently saponified; side chain ester protecting groups were transesterified but not saponified under these conditions. However, the saponification step was slow when either the peptide‐resin bond or the transesterifying alcohol was sterically hindered, and was accompanied by significant racemization.
BACKBONE TORSIONAL POTENTIAL FUNCTIONS FOR ROTATIONS ABOUT N‐Cα AND Cα‐C BONDS IN DIPEPTIDE MODEL SYSTEMS IN RELATION TO NUCLEAR MAGNETIC RESONANCE AND INFRA RED SPECTRAL DATA*
Authors: Mitra Alok K.
Published: August 2010

A comparative study of the conventional three‐fold torsional potential functions (referred to as “Old V”), the potential function having zero φ barrier and a twofold ψ‐barrier, proposed from this laboratory (“Int V”), and those developed recently in this laboratory from the study of dipeptide conformations in protein crystal structures (“New V”) has been carried out by comparing theoretically calculated and experimentally observed NH‐CαH coupling constants from n.m.r. studies and the relative occupancy of hydrogen‐bonded conformations from i.r. studies of dipeptide model systems. It is observed that no fully satisfactory agreement with both n.m.r. and i.r. data is possible in any one of the three functions. For the hydrogen bond‐disrupting solvent DMSO, (NH‐CαH) coupling constant values were predicted equally well in all three. However, only for New V are values of the constants A (= 8.62), B (= ‐2.33), C (= 1.51) as obtained by the least‐squares method in the expression J(θ) = A cos2θ+ B cos θ+ C sin2θ such that J‐values in excess of 10Hz, observed experimentally in some cases, are permissible. However, the same potential function yields unsatisfactory agreement in the case of hydrogen bond‐promoting solvent CCl4. This was interpreted to be because the potential function concerned was obtained from data in solid state where solvent interaction is absent.

In view of the lack of satisfactory agreement with the solution data for all three potential functions, it was concluded that solvent effects other than the promotion of intramolecular hydrogen bonding, which were ignored in the present study, must be taken into account for better prediction of experimental data from theoretical potential functions.
BENZYLOXYCARBONYLARGININE NITROPHENYL ESTER SALTS: 1‐HYDROXYBENZOTRIAZOLE CATALYZED ACYLATIONS OF AMINES
Authors: Glass John D. , Pelzig Michal
Published: August 2010

Benzyloxycarbonylarginine p‐nitrophenyl ester has been prepared by the p‐nitrophenyltrifluoroacetate method. The p‐nitrophenyl ester derivative was isolated as its crystalline picrate and nitrate salts. The ester salts couple with amino compounds in the presence of 1‐hydroxybenzotriazole, but decompose without acylation of amines in the absence of the 1‐hydroxybenzotriazole catalyst. Benzyloxycarbonylarginine p‐nitrophenyl ester and other activated esters of N‐α‐substituted arginine salts may be useful reagents for introduction of trypsin‐labile protecting groups into peptide fragments for purpose of polypeptide semi‐synthesis. At the same time, side reactions of such carboxyl‐activated arginine derivatives may serve as models for side reactions in the couplings of peptide fragments with arginine residues in the carboxyl‐terminal position. Peptide fragment couplings of this type may frequently be encountered in semi‐synthesis of polypeptides from tryptic fragments.
ISOLATION AND PROPERTIES OF TWO FRAGMENTS FROM PLASMIN DIGESTS OF HUMAN CHORIOMAMMOTROPIN
Authors: Li Choh Hao , Houghten Richard
Published: August 2010

Two fragments have been isolated and characterized from plasmin digests of human choriomammotropin. It was shown that these two fragments were derived from the cleavage of the Arg‐Arg (positions 133–134) and the Lys‐Gln (positions 140–141) bonds of the hormone: one has 133 amino acids and the other 51 amino acids. Both fragments were biologically and immunologically inactive.
N‐ALKYLATION OF AMINO ACID RESIDUES BY CHLOROMETHYL GROUPS
Authors: Christensen M. , Schou O.
Published: August 2010

A serious side reaction in the peptide synthesis on the Merrifield resin was observed during attempts to synthesize the TMV fragment, Asn‐Pro‐Thr‐Thr‐Ala (101–105). The side reaction is consistent with N‐alkylation of the amino groups by the residual chloromethyl groups on the resin, which lowers the total yield and complicates evaluation of monitoring data during the synthesis. It is shown that several amino acids can be N‐alkylated in different positions in the peptide chain and in different solvents. The extent of N‐alkylation is in some cases 50%.
SOLID‐PHASE SYNTHESIS OF OXYTOCIN, DESAMINOOXYTOCIN AND 4‐THR‐OXYTOCIN USING ACTIVE ESTERS IN PRESENCE OF 1‐HYDROXYBENZOTRIAZOLE
Authors: Khan Shabbir Ahmed , Sivanandaiah K.M.
Published: August 2010

Using appropriate amino acid active esters (3 eq.) in presence of HOBt* (1 eq.) and employing DPM protection for the thiol function of cysteine, a rapid synthesis of oxytocin in the solid phase has been accomplished. The DPM group has been removed by sodium‐liquid ammonia reduction since boiling TFA is ineffective. Desaminooxytocin and 4‐Thr‐oxytocin have been synthesized using lesser quantities of amino acid active esters (1.5 eq.) in presence of HOBt (1 eq.), but the durations of coupling are longer. The solid‐phase synthesis of desaminooxytocin using appropriate Boc‐amino acids in presence of DCCI in toluene medium has been described. Toluene does not exert any significant accelerating influence on the coupling rate as it does when active esters are employed.
βh‐ENDORPHIN: ANTIDIURETIC EFFECTS IN RATS
Authors: Tseng Liang‐Fu , Loh Horace H. , Li Choh Hao
Published: August 2010

Human β‐endorphin (βh‐EP) inhibits urine flow in rats. This antidiuretic effect of the peptide occurs after intravenous and intraventricular injections. Intravenously, βh‐EP is 24 times more potent than morphine. Intraventricularly, βh‐EP is 24 times more potent than morphine. Intraventricularly, βh‐EP is effective at doses (0.45 μg) which have no antidiuretic activity when injected intravenously. This fact suggests that one site of the antidiuretic action of βh‐endorphin may be in the central nervous system. Animals tolerant to morphine are also tolerant to the antidiuretic effects of βh‐endorphin.
SYNTHESES OF MONOCYCLIC AND BICYCLIC PEPTIDES OF TRYPTATHIONINE AND GLYCINE
Authors: Zanotti Giancarlo , Birr Christian , Wieland Theodor
Published: August 2010

L‐3a‐Hydroxy‐1.2.3.3a.8.8a‐hexahydropyrrolo[2,3‐b‐]‐indole‐2‐carboxylic acid (Hpi)*, obtained from L‐tryptophan by oxidation with peroxyacetic acid (Savige, 1975), after introduction of the Boc‐residue at N‐1, is coupled with various glycine peptides of S‐trityl‐L‐cysteine to give the Hpi‐peptides 6(a‐f). By treatment with absolute trifluoroacetic acid these peptides are converted by an intramolecular thiolysis to the monocyclic thioethers 7(a‐f). Two of them, 7e and 7f, can be subjected to a second cyclization by the mixed anhydride method thus yielding the bicyclic tryptathionine heptapeptide 8e and octapeptide 8f. In their structures the bicyclic molecules resemble the mushroom phallotoxins and amatoxins, respectively. They show CD spectra closely related to the naturally occurring bicyclic peptides thus indicating conformational similarities. The CD spectra of the other cyclic peptides synthesized are also presented and discussed.
COMPARATIVE TEMPERATURE‐STABILITY PROPERTIES OF MALATE DEHYDROGENASES FROM SOME THERMOPHILIC FUNGI*
Authors: WALI ANUPAM S. , MATTOO AUTAR K. , MODI VINOD V.
Published: August 2010

Temperature‐activity and temperature‐stability relationships of malate dehydrogenases from eight thermophilic fungi were determined. Temperature optima for maximum activity of the enzymes varied between 50° and 60° and the Arrhenius plots were linear between 5° and 50°. The energies of activation ranged from 2.1 K cal/mol for the enzyme from Sporotrichum thermophile to 9.1 K cal/mol for that from Penicillium duponti. Heat inactivation kinetics at 50° revealed heat lability of the enzyme from most of the thermophilic fungi. The t1/2's (min) were < 10 for the enzymes from P.duponti, S.thermophile and Thermoascus aurantiacus; < 30 for those from Chaetomium thermophile var. coprophile, H.lanuginosa and C.thermophile var. dissitum; and > 30 for those from Mucor pusillus and H.insolens. Salts of Na+, K+ and NH4+, and citrate protected the enzymes from H.lanuginosa, C.thermophile var. dissitum and M.pusillus against heat inactivation.
THE PURIFICATION OF PEPTIDES BY PARTITION CHROMATOGRAPHY BASED ON A HYDROPHOBICITY SCALE*
Authors: YAMASHIRO DONALD
Published: August 2010

A study of the efficiency of partition chromatography for the purification of peptides as a function of structure has been undertaken. A series of 19 omission analogs of camel β‐endorphin and of some of its partial sequences have been synthesized with each analog missing only a single amino acid. Their chromatographic properties have been examined with use of the Martin hypothesis and the RM concept, and a hydrophobicity scale for the amino acid side chains was obtained. To a first approximation a correlation with the Tanford hydrophobicity scale for amino acids was found. A decrease in hydrophobicity has been observed with increasing chain length and is discussed in terms of column efficiencies required for the purification of synthetic peptides.
HALOACETYL GROUPS AS REVERSIBLE PROTECTION OF THE AMINO FUNCTION: CLEAVAGE WITH 2‐AMINOTHIOPHENOL
Authors: GLASS JOHN D. , PELZIG MICHAL , PANDE CHANDRA S.
Published: August 2010

Haloacetylamino acids and haloacetyl peptides react rapidly with 2‐aminothiophenol in weakly alkaline media to yield 2‐aminothiophenoxyacetyl derivatives. These intermediates are subject to acidolysis under mild conditions with release of free amino acids or peptides. With this mild method for removal of the haloacetyl group N‐haloacetoxysuccinimide derivatives, which rapidly and specifically acylate amino groups of polypeptides in aqueous solutions, become promising reagents for the reversible protection of amino groups. The chloro‐acetylation of amino groups in lima bean trypsin inhibitor and the quantitative removal of the chloroacetyl groups demonstrate the applicability of the method for polypeptides. The haloacetyl group also serves an analytical function in that treatment of a completely or partially haloacetylated polypeptide with cysteine forms one carboxymethylcysteine residue per haloacetyl group in the polypeptide derivative. Carboxymethylcysteine is readily measured by amino acid analysis of acid hydrolysates.

Approaches to further improvement of conditions for removal of haloacetyl groups are discussed and potential applications of the general chemistry of 2‐haloacids to modern polypeptide chemistry are outlined.
SOLID PHASE SYNTHESIS WITHOUT REPETITIVE ACIDOLYSIS
Authors: MEIENHOFER JOHANNES , WAKI MICHINORI , HEIMRE EDGAR P. , LAMBROS THEODORE J. , MAKOFSKE RAYMOND C. , CHANG CHI‐DEU
Published: August 2010

The utility of repetitive nonhydrolytic base cleavage of α‐amino protective groups in solid phase peptide synthesis is shown by a preparation of the model tetrapeptide leucyl‐alanyl‐glycyl‐valine on a p‐benzyloxybenzyl ester polystyrene–1% divinylbenzene resin support. Nα‐9‐Fluorenylmethyloxycarbonyl (Fmoc: Carpino & Han, 1970, 1972) amino acids were coupled by the symmetrical anhydride procedure, followed by Fmoc group cleavage using 50% piperidine in methylene chloride. Quantitative removal of the Fmoc‐tetrapeptide from the solid support was effected by treatment with 55% trifluoroacetic acid in methylene chloride. Homogeneous free tetrapeptide was obtained in 87% overall yield. The procedure is proposed to offer advantages over present solid phase methods which use acidolysis for repetitive α‐amino group deblocking.
[21 ‐ ARGININE ‐ A] INSULIN
Authors: FERDERIGOS NICOLAOS , COSMATOS ALEXANDROS , FERDERIGOS ANGELIKI , KATSOYANNIS PANAYOTIS G.
Published: August 2010

An analog of sheep insulin which differs from the parent molecule in that the C‐terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S‐sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S‐sulfonated B chain of bovine (sheep) insulin yielded [Arg21‐A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21‐A] sheep insulin shows potencies of 10.5–12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23–25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge‐ and hydrogen bond‐forming interactions which are critical in the biological activity of the hormone. Although the [Arg21‐A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the abovementioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.
ENZYMATIC DETERMINATION OF PYRIDOXAL 5‘‐PHOSPHATE WITH γ‐CYANOAMINOBUTYRIC ACID APOSYNTHASE
Authors: ABE KAZUNORI , RESSLER CHARLOTTE
Published: August 2010

A rapid alternative method is presented for the determination of pyridoxal 5′‐phosphate (pyridoxal‐P). The method involves the colorimetric analysis of thiocyanate liberated from S‐cyanohomocysteine (Hcy(CN)) in the presence of cyanide when catalyzed by the pyridoxal‐P dependent enzyme, γ‐cyano‐α‐aminobutyric acid (γ‐CNabu)‐synthase (Hcy(CN) thiocyano‐lyase [adding CN]). The rate of formation of thiocyanate is determined by the increase in absorbance at 470nm on treatment of the enzymatic reaction mixture with FeCl3.
POLY‐N‐ACRYLYLPYRROLIDINE A NEW RESIN IN PEPTIDE CHEMISTRY
Authors: SMITH CLARK W. , STAHL GLENN L. , WALTER RODERICH
Published: August 2010

Entirely beaded poly‐N‐acrylylpyrrolidine‐co‐bisacrylyl‐1, 2‐diaminoethane‐co‐N‐acrylyl‐1, 6‐diaminohexane‐HCl(PAP), a new resin on which to perform peptide chemistry, has been prepared by reverse phase suspension polymerization in quantitative yield. In addition to being a superior support to polystyrene, albeit readily adaptable to current techniques of peptide synthesis, its versatility has been further extended by the introduction and use of new peptide‐to‐polymer linking groups, which allow the use of the bidirectional approach to peptide chemistry. One such linkage, which connects the side chain of cysteine to PAP via an acid resistant S‐carbamoyl bond, was used in a bidirectional synthesis of deamino‐oxytocin. PAP solvates and swells in solvents with wide‐ranging polarities, including aqueous media. Thus, peptide coupling reactions were performed in organic media of high and of low polarity as well as in aqueous solution.
SYNTHESES AND BIOLOGICAL ACTIVITIES OF METHOXININE‐ANALOGUES OF THE C‐TERMINAL OCTAPEPTIDE OF CHOLECYSTOKININ‐PANCREOZYMIN
Authors: GILLESSEN DIETER , TRZECIAK ARNOLD , MÜLLER RITA K. M. , STUDER ROLF O.
Published: August 2010

The C‐terminal octapeptide of cholecystokinin‐pancreozymin and its analogues in which the methionine residues were successively replaced by methoxinine have been synthesized. When these analogues were tested for their gallbladder‐contraction activity in an in vitro system the following relative potencies were observed: [Mox3]‐CCK‐PZ 8 (95%), [Mox6]‐CCK‐PZ 8 (5%) and [Mox3, Mox6]‐CCK‐PZ 8 (2%).
SOLUBLE DI‐ AND AMINOPEPTIDASES IN ESCHERICHIA COLI K‐12 Dispensible Enzymes
Authors: HERMSDORF CAROL L. , SIMMONDS SOFIA , SAUNDERS ALDA
Published: August 2010

As part of a study of the peptidase content of Escherichia coli K‐12, two peptidase‐deficient amino acid auxotrophs isolated and characterized by Miller as pepD‐ (strain CM17) and pepD‐ pepN‐ pepA‐ pepB‐ pepQ‐ (strain CM89) were examined for the presence of several peptidases previously obtained from strain K‐12 in this laboratory. The soluble fraction of each mutant was found to lack the broad‐specificity strain K‐12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K‐12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide‐specific amino‐peptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome‐bound aminopeptidase I of strain K‐12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD‐, pepB‐, pepN‐, and pepA‐ mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di‐ or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K‐12 and produced smaller cell‐yields than those produced by strain K‐12.
SORGHUM PROTEINASE INHIBITORS
Authors: KUMAR P.M. HARISH , VIRUPAKSHA TUMKUR K. , VITHAYATHIL PAUL J.
Published: August 2010

Investigations have been carried out on the complex formed between sorghum Inhibitor III and α‐chymotrypsin by physico‐chemical methods. An apparent dissociation constant (Ki) of 4.0 times 10‐8 M has been calculated for the complex. This enzyme‐inhibitor complex was isolated by gel filtration on Sephadex G‐75 and a molecular weight of 48,000 was estimated for the complex. The formation of the complex was accompanied by spectral changes in the 270–300 nm region of the spectrum. Preliminary evidence suggests that the sorghum Inhibitor III is structurally altered when it is incubated with α‐chymotrypsin. Catalytically inactive derivative of α‐chymotrypsin and trypsin, as well as their zymogens, did not interfere with the activity of the sorghum inhibitor towards the native enzymes. Sorghum Inhibitor I was shown to be a ‘double‐headed’ inhibitor since it inhibits both trypsin and α‐chymotrypsin independently.
MICROHETEROGENEITY OF HUMAN KININOGEN BY ISOELECTRIC FOCUSING AND CROSSED IMMUNOELECTROPHORESIS
Authors: TURPEINEN URSULA , HAMBERG ULLA
Published: August 2010

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G‐200 (Kav0.34) followed by DEAE‐chromatography according to earlier established methods. Further purification was performed with specific Sepharose‐antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH‐gradients 3.5–10, 4–6 and 3.5–5. Kininogen components were determined by single radial immuno‐diffusion against monospecific anti‐human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pl 4.5; the respective focusing ranges were pI 4.4–4.7 (60–80%) and pI 4.3–4.6 (92%). The results were verified by crossed immuno‐electrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and α2HS‐glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5–5.0 and four α2HS components at pI 4.2–4.6.
MALEYLATION AND PH‐DEPENDENCE OF STREPTOMYCES GRISEUS PROTEASE B
Authors: SHINAR SHULAMIT , GERTLER ARIEH
Published: August 2010

The enzymatic activity of Streptomyces griseus protease B (SGPB) was measured over pH range 8.4–11.5 using a specific new, chromophoric substrate N‐succinyl‐glycyl‐glycyl‐l‐phenylalanine p‐nitroanilide. It was found that the activity is dependent on ionization of a single group with apparent pK = 10.84, possibly lysine‐125. Maleylation of the ∍‐amino group of this lysine was linearily associated with the loss of enzymatic activity.

It is therefore suggested that the electrostatic interaction between the side chain of lysine‐125 and the α‐carboxyl group of the the C‐terminal tyrosine is crucial to the active conformation of the enzyme. In contrast the maleylation of the α‐amino group of the N‐terminal isoleucine was rapid but could not be correlated to the loss of activity.
FACILE THIOLYTIC REMOVAL OF THE o‐NITROPHENYLSULPHENYL AMINO‐PROTECTING GROUP
Authors: STERN MEIR , WARSHAWSKY ABRAHAM , FRIDKIN MATI
Published: August 2010

2‐Mercaptopyridine was used to effect the selective, mild and efficient cleavage of the o‐nitrophenylsulphenyl amino‐protecting group from several amino acids and peptides. By utilization of this reagent a stepwise synthesis of the tetrapeptide Thr‐Lys‐Leu‐Arg([Leu3]tuftsin) was successfully achieved. The potential use of 2‐mercaptopyridine in mechanized peptide synthesis via the polymeric reagents approach is discussed.
SOLID PHASE SYNTHESIS OF 13‐LY SINE‐APAMIN, 14‐LYSINE‐APAMIN AND THE CORRESPONDING GUANIDINATED DERIVATIVES
Authors: SANDBERG BENGT E.B.
Published: August 2010

In order to study the importance of arginine residues 13 and 14 in apamin, the bee venom neurotoxin, four analogues, [Lys13]‐apamin, [Lys14]‐apamin, [Har4, Har13]‐apamin and [Har4, Har14]‐apamin were synthesized and tested with respect to their neurotoxicity. The two lysine‐apamins were prepared by the solid phase method on benzhydrylamine resins. Before oxidation to disulphides, the (S‐Acm)4‐peptides were isolated and characterized. Portions of the purified lysine peptides were converted to homoarginine analogues by guanidination. The four apamin analogues were lethal, but the lethal doses differed significantly. The results demonstrate that the arginine residue at position 14 is more important for the high toxicity than is the one at position 13. The circular dichroism [CD) spectrum of [Lys13]‐apamin was identical with that of apamin itself, whereas the spectrum of [Lys14]‐apamin showed certain deviations.
SYNTHESIS OF Nα‐FORMYL‐MET‐LEU‐PHE‐OH: AN INDUCER OF CHEMOTAXIS IN PERITONEAL POLYMORPHONUCLEAR NEUTROPHILS
Authors: DAY ALAN R. , FREER RICHARD J.
Published: August 2010

A chemotactic peptide CHO.Met.Leu.Phe.OH has been synthesized classically using the mixed anhydride procedure. The formyl group was introduced by coupling formic acid in the presence of dicyclohexylcarbodiimide to the partially protected tripeptide. The final product was obtained by treatment of the intermediate CHO.Met.Leu.Phe.OBzl with hydrogen fluoride. The ED50 of the peptide in the Boyden chamber assay was 7 × 10‐11 M; in the lysozyme release assay 2.4 × 10‐10 M and in the β‐glucuronidase release assay 2.6 × 10‐10 M. In a radioreceptor assay the ID50 of the peptide was 3.3 × 10‐10 M.
INDUCED HELICAL CONFORMATION OF IONIC POLYPEPTIDES BY PHOSPHOLIPIDS SOLUBILIZED IN A NONIONIC SURFACTANT SOLUTION
Authors: SHIRAHAMA KEISHIRO , YANG JEN TSI
Published: August 2010

Phosphatidylcholine that has been solubilized in hexadecylpoly (oxyethylene) ether can induce a coil‐to‐helix transformation for poly(l‐glutamic acid) in neutral solution. Similarly, solubilized phosphatidylserine promotes a helical conformation of poly(l‐lysine) at neutral pH through complex formation. The mixed micelles of a phospholipid and a nonionic surfactant are thermodynamically stable and do not separate on standing. The nonionic surfactant here has no effect on the conformation of the two polypeptides.
EFFECTS OF d AND l AMINO ACID RESIDUES IN LINEAR PEPTIDES ON CARBON‐13 NUCLEAR MAGNETIC RESONANCE PARAMETERS
Authors: DESLAURIERS ROXANNE , SMITH IAN C.P. , SOMORJAI R.L. , RALSTON E. , ORLOWSKI R.C. , WALTER RODERICH
Published: August 2010

The 13C spectra of the linear tripeptidyl diastereoisomers, Gly‐Gly‐Leu, Gly‐Gly‐d‐Leu, Leu‐Gly‐Gly, d‐Leu‐Gly‐Gly, Ala3, Ala‐Ala‐d‐Ala, Ala‐d‐Ala‐Ala, Val3, and Val‐Val‐d‐Val are very similar or even identical at pH meter readings of 1.0, 7.0 and 12.0 in D2O. The spectra of Pro‐Leu‐Gly‐NH2 and Pro‐d‐Leu‐Gly‐NH2 likewise show only minor differences in 13C chemical shifts (less than 0.4 p.p.m.) under similar conditions. This contrasts significantly with previous findings comparing 13C chemical shifts of cyclo (Pro‐Leu) and cyclo (Pro‐d‐Leu) where major differences in chemical shifts were observed for both residues due to differences in conformational constraints present in these cyclic prolinecontaining peptides.

The least‐squares fit of spin‐lattice relaxation times (T1) for Pro‐Leu‐Gly‐NH2 and Pro‐d‐Leu‐Gly‐NH2 show that it is not possible to fit all the T1 values to a unique and rigid structure whether folded or extended. The glycyl residue undergoes enhanced motion when compared with the prolyl and leucyl residues. Internal motion must be postulated within the proline ring and for the CH3 groups of leucine.
PEPTIDE–WATER ASSOCIATION IN PEPTIDE CRYSTALS
Authors: YANG CHIA‐HSI , BROWN JOE N. , KOPPLE KENNETH D.
Published: August 2010

The structures of 37 peptide crystals, containing 78 water‐peptide hydrogen bonds and 77 other hydrogen bonds involving water, were surveyed to identify the geometry of peptide backbone hydration. In the sample, hydration of peptide carbonyl occurred more frequently than hydration of peptide N–H. The most probable value of the C'=0. O water angle was near 138°, considerably greater than the 120° to the axis of a lone electron pair on the carbonyl oxygen. Associated water oxygens tended to be in the plane of the peptide bond, but not restricted to it. Cyclic association with water, to form 10‐membered rings using Ni–H and Ci + 1 =0 atoms, was common in glycine‐containing cyclic hexapeptides. The distribution of angles between two hydrogen bonds at a single water molecule, as defined by the three nonhydrogen atoms involved, was centered near the tetrahedral angle.
SYNTHESIS, RESOLUTION AND CHARGE‐DONOR PROPERTIES OF SIX TRYPTOPHAN ANALOGUES
Authors: RAJH HUBERTUS M. , UITZETTER JOSEPH H. , WESTERHUIS LAMBERTUS W. , van den DRIES CORNELIS L. , TESSER GODEFRIDUS I.
Published: August 2010

In order to investigate the special function of tryptophan in peptide hormones, six tryptophan analogues have been synthesized, in which structural resemblance has been grossly retained and potentially essential properties have only partially been varied. The l‐enantiomers have been isolated after enzymatic digestion of racemic dipeptide derivatives, and charge‐transfer properties of the compounds have been studied.
EFFECT OF SODIUM DODECYL SULPHATE ON THE 12S FRACTION OF MUSTARD PROTEINS (B. JUNCEA)
Authors: RAO A. GURURAJ , RAO M.S. NARASINGA
Published: August 2010

The effect of SDS* on the 12S protein fraction of mustard seed (B. juncea) has been followed by the techniques of ultracentrifugation, gel filtration, gel electrophoresis, viscosity, ultraviolet difference spectra and fluorescence spectra. At low concentrations of SDS, up to 0.1%, both aggregation and dissociation of the protein occurs. Only dissociation occurs at higher SDS concentrations and is complete at 0.5% SDS. Viscosity increases sharply up to 0.15% SDS, remains constant between 0.15 and 0.30% and then increases markedly again, SDS induces also difference spectra with minima at 280, 288 and 295 nm. Fluorescence emission intensity increases at SDS concentrations < 0.05% and quenching occurs at higher SDS concentrations. The results suggest that SDS causes association, dissociation and denaturation of the protein molecule.
BOVINE NEUROPHYSIN LIPID COMPLEX Their Isolation, Characterization and Reaggregation
Authors: SAWADA YOSUKE , AUDHYA TAPAN K. , WALTER RODERICH
Published: August 2010

A lipid‐containing neurophysin fraction was isolated and purified from bovine posterior pituitary glands by acid extraction and affinity chromatography on a heparin‐Sepharose 4B column. This lipid‐rich fraction was found to be composed of noncovalent aggregates of neurophysin proteins and phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin. The lipid‐containing neurophysin was delipidated by treatment with chloroform‐methanol. The resultant apoproteins were characterized as bovine neurophysin‐I and ‐II by immunological and electrophoretic procedures. Conditions were developed for the reaggregation of purified bovine neurophysin‐I and ‐II with lipids extracted from bovine posterior pituitary and hypothalamus and with synthetic lecithin. The resultant neurophysin lipid complexes have been shown to band upon isopycnic centrifugation at densities different from those of the respective purified bovine neurophysins.
SHORT IN VITRO HALF‐LIFE OF THYMOPOIETIN 32–36 PENTAPEPTIDE IN HUMAN PLASMA
Authors: TISCHIO JOHN P. , PATRICK JAMES E. , WEINTRAUB HOWARD S. , CHASIN MARK , GOLDSTEIN GIDEON
Published: August 2010

Thymopoietin32–36(TP5) is a synthetic pentapeptide that has the biological activity of its parent molecule, the 49 amino acid thymic hormone thymopoietin. Tritiated thymopoietin32–36(3H‐TP5) was prepared by reductive tritiation of dibromotyrosyl‐TP5. The stability of3H‐TP5 in human plasma was studied by analyzing samples by thin‐layer chromatography at different time points and quantitating the radioactivity associated with TP5 (Arg‐Lys‐Asp‐Val‐Tyr) and its tyrosyl‐containing breakdown products (Lys‐Asp‐Val‐Tyr, Asp‐Val‐Tyr, Val‐Tyr, Tyr). In plasma (but not in saline) the pentapeptide was rapidly degraded (apparent t1/2‐30 seconds) with the corresponding appearance of radioactivity associated with the other tyrosyl‐containing reference compounds. These data imply that the pentapeptide, which is activein vivo,may rapidly trigger changes in responsive cells; sustained circulating levels may not be required for activity.
FORMATION OF AMINOSUCCINYL PEPTIDES DURING ACIDOLYTIC DEPROTECTION FOLLOWED BY THEIR TRANSFORMATION TO PIPERAZINE‐2, 5‐DIONE DERIVATIVES IN NEUTRAL MEDIA
Authors: SCHÖN ISTVÄN , KISFALUDY LAJOS
Published: August 2010

Prolonged acidic treatment of Boc‐Leu‐Asp(OBut)‐Phe‐NH2 with 4N HCl in acetic acid resulted in H‐[Leu‐Asc‐Phe‐NH2] HCl(Asc, aminosuccinyl), which transformed partially to cyclo[Leu‐Asp(Phe‐NH2)] during its purification by column chromatography on silica gel with a mixture of ethyl acetate/pyridine/acetic acid/water = 60:20:6:11, i.e. in neutral medium. Examination of the imide formation was extended to different reaction conditions (no imide derivative was detected in trifluoroacetic acid), to several protected derivatives of L‐aspartyl‐L‐phenylalaninamide and to tripeptides containing an aspartyl residue in the middle position. It was clearly demonstrated that in strongly acidic media the imide derivatives are directly formed from the aspartyl peptides containing a free β‐carboxyl group.

The influence of the C‐terminal residue was greater than the N‐terminal on both the rate of formation of the imide and its further transformation to piperazine‐2,5‐dione derivative. In aqueous ethanol the X‐Asc‐Y‐NH2 (X, Pro, Leu; Y, Gly, Ala, Val, Phg, Phe) containing N‐terminal proline are more readily transformed to piperazine‐2,5‐dione derivatives, but compared to simple proline dipeptides the rate of this transformation is relatively slow because of the crowdedness of the tricyclic transitional state.
EMPIRICAL CALCULATIONS ON CYCLIC DIPEPTIDES
Authors: GENEST M. , PTAK M.
Published: August 2010

Empirical conformational energy calculations were carried out for the dipeptides cyclo‐(L‐Asp‐L‐His) and cyclo‐(L‐Glu‐L‐His). The side chain conformations have been studied for various DKP ring structures, demonstrating that distortions can modify the stability of some conformations by favouring peptide backboneside chain or side chain ‐ side chain interactions. The carboxylic side chain of Asp and Glu residues can induce specific interactions with the imidazole ring or the peptide bond of the DKP ring which are not found for Ser or Thr. The necessity of including solute‐solvent interactions to account for a more realistic conformational behaviour is discussed.
A NOVEL SYNTHESIS OF 2‐THIOETHER DERIVATIVES OF TRYPTOPHAN
Authors: SAVIGE WALTER E. , FONTANA ANGELO
Published: August 2010

The reaction of L‐3a‐hydroxy‐1,2,3, 3a, 8,8a‐hexahydropyrrolo[2,3‐b]indole‐2‐carboxylic acid (Hpi) with methanethiol, ethanethiol, mercaptoethanol or 3‐mercaptopropionic acid in warm aqueous acetic acid gives the corresponding 2‐thioether derivatives of tryptophan in 50–80% yield (based on Hpi). Better yields may be obtained in 25% trifluoroacetic acid at room temperature. Cysteine reacts with Hpi to give the double amino acid 2‐(L‐3‐alanylthio)‐L‐tryptophan (tryptathionine), which is a constituent of the highly poisonous cyclopeptides of Amanita phalloides, such as phalloidin. Reaction of a moderate excess of Hpi with cysteine‐SH groups of a tripeptide (glutathione) and a protein (reduced ribonuclease) has also been effected, giving the respective S‐tryptophanylated peptide or protein. In both cases, reaction occurred specifically with the ‐SH groups of cysteine and virtually quantitative covalent binding of tryptophan was verified. The extent of the reaction is easily quantitated by spectrophotometry or by amino acid analysis of the content of oxindolylalanine in the hydrolysate with hot 3‐N p‐toluenesulfonic acid of the S‐tryptophanylated peptide or protein. The reaction should be useful in the field of peptide synthesis, providing a simple method for establishing a cross‐link between tryptophan and cysteine, as a basic step in the chemical synthesis of toxic peptides of Amanita phalloides.
ENOLASE FROM BACILLUS STEAROTHERMOPHILUS
Authors: SALVATO B. , BOCCÙ E. , GRANDI C. , FONTAAN A. , VERONESE F.M.
Published: August 2010

Enolase from Bacillus stearothermophilus was homogeneous in the ultracentrifuge and had the hydrodynamic properties of a globular protein with a molecular weight of 3.51 times 105 (sedimentation‐diffusion) and 3.60 times 105 (Archibald approach to equilibrium). Electrophoretic and gel filtration measurements in presence of dissociating agents indicated that the enzyme consists of eight apparently identical polypeptide chains of molecular weight of 47 000. The denaturation of enolase has been studied as a function of urea concentration. A broad transition curve was observed, the enzyme in 8 M urea being inactive and dissociated into monomers. The far‐ultraviolet circular dichroism spectrum of enolase in 8 M urea solution indicates extensive unfolding of the globular structure. Accordingly, fluorescence emission measurements indicate that the tryptophanyl residues of the denatured enolase are largely exposed to the aqueous solvent medium. The enzyme can be reversibly denatured by 8 M urea and the enzymatic activity then recovered by dilution 1:100 of the denatured enzyme into buffer or by removing urea by dialysis. Under suitable conditions and in the presence of Mg2+‐ions, 90–100% of original activity was recovered and the renatured enzyme was indistinguishable from the native one, as judged from several enzymological and physicochemical criteria, including molecular weight. The far‐ultraviolet circular dichroism spectrum and the fluorescence emission properties of the native enzyme were regained very rapidly (less than 1 min), while enzymatic activity was recovered much more slowly. Octameric enzyme species were obtained on refolding the denatured enzyme at temperatures between 4 and 55.
SYNTHESIS AND ANALGESIC ACTIVITY OF HUMAN β‐ENDORPHIN ANALOGS SUBSTITUTED AT POSITIONS 17, 18, OR 19
Authors: BLAKE JAMES , TSENG LIANG‐FU , LI CHOH HAO
Published: August 2010

Three peptide analogs of β‐endorphin which are substituted in positions 17, 18 or 19 have been synthesized and their analgesic potencies have been measured by the tail‐flick method in mice. The results showed that the replacement of Phe‐18 or Lys‐19 by alanine reduced the potency to 15% whereas the replacement of Leu‐17 by alanine reduced the analgesic potency to 68%.
ISOLATION AND CHARACTERIZATION OF β‐LIPOTROPIN FROM FIN WHALE PITUITARY GLANDS
Authors: KAWAUCHI HIROSHI , CHUNG DAVID , LI CHOH HAO
Published: August 2010

The isolation and characterization of β‐lipotropin from fin whale (Balaenoptera physalus) pituitary glands are described. The proposed primary structure is also presented. Fin whale β‐LPH exhibited an identical lipolytic activity when compared with human hormone.
MODIFICATIONS OF SOLID PHASE PEPTIDE SYNTHESIS TO OBTAIN HOMOGENEOUS OLIGOPROLINES IN HIGH YIELD
Authors: AINPOUR PARVIZ ROBERT , WICKSTROM ERIC
Published: August 2010

When oligoprolines containing a [3H] proline in the second residue from the carboxyl terminus are synthesized by the standard solid phase method using stepwise addition of prolines there appears, in addition to the oligoprolines, a steady increase of ninhydrin‐negative, but radioactive, impurities. These impurities were found to be N‐trifluoroacetyl oligoprolines, and a substantial amount of diketopiperazine was present at the diproline stage. The trifluoroacetyl blockage of the N‐termini was almost complete by the time 14 residues were added. To eliminate these side reactions, a block of Boc[3H]Pro2 was coupled to prolyl‐resin in order to avoid the diprolyl‐resin stage, and 25% CF3CO2H in CHCl3 was replaced by 4N HCl in dioxane as the deprotecting reagent. These changes eliminated the formation of diketopiperazine and trifluoroacetyl impurities, but still a steady increase of ninhydrin‐negative, but radioactive, impurity was seen, reaching 22% after three couplings. These side reactions were traced to the neutralization step, and in order to avoid them it became necessary to eliminate the neutralization step, and replace it by incremental additions of Et3N during the coupling step. As a result of these modifications, we now obtain homogeneous length oligoprolines upon cleavage from the resin, with no further purification, when analyzed by reversed‐phase liquid chromatography.
N‐TERMINAL AMINO ACID SEQUENCE OF PROALBUMIN FROM INBRED BUFFALO RATS
Authors: MILLERSHIP ANNE , EDWARDS KAYLENE , CHELLADURA MOHANATHASAN , DRYBURGH HEIDE , INGLIS ADAM S. , URBAN JÖRG , SCHREIBER GERHARD
Published: August 2010

The sequence of radioactively labelled amino acids at the N‐terminus of proalbumin was determined by automated Edman‐degradation. [3H] Valine, [3H] phenylalanine or [14Carginine was incorporated into protein in vivo for a time period of 10 min after injection. Since albumin remains unlabelled during this time period (Urban et al., 1976), separation of proalbumin and albumin was not required for this work. Hence, compared to previous methods, a shorter purification procedure could be used which increased the yield of anti‐albumin‐precipitable protein and reduced the risk of proteolysis.

Microsomes were prepared from livers removed 10 min after injection of the radioactively labelled amino acids. A buffer extract of the acetone‐dried powder from these microsomes was chromatographed on DEAE‐cellulose. All protein obtained after chromatography which could be precipitated with antiserum to serum albumin was isolated by immunoprecipitation and subsequent separation of the antigen‐antibody complex. The sequence of radioactive amino acids in this antigen preparation suggests that about 20–25% of proalbumin possessed at the N‐terminus the pentapeptide sequence X‐Val‐Phe‐Arg‐Arg‐ whereas 75–80% contained the hexapeptide sequence Arg‐X‐Val‐Phe‐Arg‐Arg‐.