bluuebunny/crossref_metadata_2025 huggingface dataset:
DISSOCIATION, AGGREGATION AND DENATURATION OF SESAME α‐GLOBULIN IN UREA AND GUANIDINE HYDROCHLORIDE SOLUTIONS*
Authors: Prakash V. , Nandi P. K.
Published: August 2010

The effect of urea and GuHCl on the major protein of sesame seed (Sesamum indicum L.), α‐globulin, has been investigated by turbidity, sedimentation velocity, viscosity, difference spectra and fluorescence spectral measurements. The protein undergoes dissociation, aggregation and denaturation in the presence of the above denaturants. There is a critical concentration of the denaturant where aggregation is maximum. Both denaturation and aggregation are lower in buffers of high ionic strength. Dissociation and aggregation have been explained by considering two types of subunits present in the protein molecule, one leading to smaller sedimenting component and the other producing the aggregate. The amino acid analysis shows that the aggregated fraction is rich in aliphatic amino acid residues. The endothermic nature of the aggregation process has been considered to arise from hydrophobic interaction of aliphatic side chains of the relevant subunits. The protein exists in a more denatured state in GuHCl than in urea solution.
COACERVATION OF SEQUENTIAL POLYPEPTIDE MODELS OF TROPOELASTIN
Authors: Rapaka R. S. , Urry D. W.
Published: August 2010

Syntheses of two sequential polytctrapeptide models, H‐(Val‐Ala‐Pro‐Gly)n‐Val‐OMe and H‐(Val‐Pro‐Gly‐Gly)n‐Val‐OMe via the p‐nitrophenyl ester method are described. The p‐nitrophenyl ester method gave high yields (85%–100%) of large molecular weight polymers. H‐(Val‐Pro‐Gly‐Gly)n‐Val‐OMe exhibited the interesting property of coacervation on raising the temperature of aqueous solutions while H‐(Val‐Ala‐Pro‐Gly)n‐Val‐OMe precipitates irreversibly under similar conditions.

Whereas non‐dialyzed lower molecular weight polymers of H‐(Val‐Pro‐Gly‐Gly)n‐Val‐OMe with n = 8 to 40 did not coacervate, but did show a transition to increased intramolecular order on raising the temperature of aqueous solutions above 50°C, the dialyzed higher molecular weight polymer, n > 40, does coacervate beginning at about 50°C. This demonstrates the molecular weight dependence of coacervation and also suggests the importance to coacervation of side chain interactions in the Val‐Pro sequence. The increase in intramolecular order, seen as the formation of a 14‐atom hydrogen‐bonded ring, occurs simultaneously with the Val‐Pro hydrophobic side chain association.
CIRCULAR DICHROISM STUDIES ON NATIVE AND PHENYLMETHANESULFONYL‐MESENTERICOPEPTIDASE
Authors: Genov Nicolay , Shopova Maria
Published: August 2010

At neutral pH the far ultraviolet circular dichroism (CD) spectrum of alkaline mesentericopeptidase is dominated by two negative bands: at 208 and 220 nm. The near ultraviolet CD spectrum is characterized by a large negative band at 277nm, a shoulder near 286nm and a small positive band at 297nm. The introduction of phenylmethanesulfonyl (PMS) group on the serine residue at the active site does not alter the intensity and the wavelength position of the bands.

In contrast to the other alkaline bacterial proteases, which are remarkably resistant to denaturation by urea, mesentericopeptidase was unfolded in the presence of this reagent at neutral pH. The denaturation reactions in the presence of 9.5 M urea of both native and PMS‐mesentericopeptidase followed first order kinetics. The introduction of PMS‐group on the active‐site serine lowered the conformational stability of the enzyme. In the first 3h PMS‐mesentericopeptidase was denaturated 2.5 times faster than native protease.

In the presence of 9.5 M urea at natural pH alkaline mesentericopeptidase lost its catalytic activity. The loss of enzymatic activity correlated with the unfolding of the protein molecule and also followed first‐order kinetics.
STUDIES ON POLYPEPTIDES
Authors: Voskuyl‐Holtkamp Ingrid , Schattenkerk Cecile
Published: August 2010

A description is given of the synthesis by fragment condensation of the peptide Gly‐Glu‐Ser‐Arg‐Glu‐Ser‐Ser‐Ala‐Asp‐Lys‐Phe‐Lys‐Arg‐Gln‐His‐Met‐Asp‐Thr‐Glu‐Gly‐Pro‐Ser‐Lys corresponding to the 1–23 amino acid sequence of rat pancreatic ribonuclease. This rat peptide combined with bovine S‐protein yields a fully active ribonuclease S' analogue.
STUDIES OF THE INTERACTION OF BOVINE NEUROPHYSIN‐II WITH [1‐HEMI‐d‐(3‐13 C)CYSTINE] OXYTOCIN AND [1‐HEMI‐(3‐13 C)CYSTINE] OXYTOCIN
Authors: DESLAURIERS ROXANNE , SMITH IAN C.P. , STAHL GLENN L. , WALTER RODERICH
Published: August 2010

The interactions of [1‐hemi‐(3‐13C)cystine]oxytocin and [1‐hemi‐D‐(3‐13C) cystine]oxytocin with bovine neurophysin‐II have been studied in aqueous solution under various conditions of pH using carbon‐13 (13C) nuclear magnetic resonance spectroscopy (n.m.r.). We have monitored changes in 13C chemical shifts (δ), spin‐lattice relaxation times (T1) and linewidths (πT2*)‐1 of the [1‐hemi‐(3‐13C)cystine] oxytocin as a function of the concentration of added neurophysin. [1‐Hemi‐d‐(3‐13C)cystine]oxytocin does not interact with neurophysin and shows no changes in the above spectral parameters as a function of increasing protein concentrations. The 13C chemical shifts have been monitored as a function of pH for both 13C‐enriched, diastereoisomeric oxytocin analogs in the presence and absence of neurophysin. The midpoints of the pH‐titration curves for [1‐hemi‐(3‐13C)cystine]oxytocin and [1‐hemi‐d‐(3‐13C) cystine]oxytocin in the absence or presence of neurophysin are 6.2 and 5.5, respectively. T1 values are independent of pH for both peptides in free solution, and the rotational correlation times (tc) obtained for the peptides are of the order of magnitude expected from the Stokes‐Einstein relation (5.0 times 10‐10 sec rad‐1). T1 values decrease and linewidths increase for [1‐hemi‐(3‐13C)cystine]oxytocin in the presence of neurophysin. Fast‐exchange on the n.m.r. time scale occurs at low pH. Only broad lines can be detected at neutral pH rendering 13C T1 measurements difficult. It appears that increasing the concentration of neurophysin in the presence of [1‐hemi‐(3‐13C)cystine]oxytocin leads to aggregation of the system as judged by the 13C T1 results and corresponding rotational correlation times. In light of the significant changes in T1 values and small changes in δ values, it would seem that T1 measurements are more accurate monitors of peptide‐protein interactions than are 13C chemical shifts when the interaction of the peptide with the protein does not involve any changes in steric constraints of the peptide being monitored.
ENZYMATIC METHYL ESTERIFICATION OF PITUITARY POLYPEPTIDES
Authors: KIM SANGDUK , LI CHOH HAO
Published: August 2010

Methyl esterification of pituitary polypeptides by protein methylase II (S‐adenosylmethionine:protein‐carboxyl O‐methyltransferase, EC. 2.1.1.24) has been investigated. Ovine lutropin and adrenocorticotropin** (α1–39‐ACTH) were found to be good methyl acceptor substrates, followed by β‐lipotropin. While the α‐subunit of lutropin had nearly equal the methyl accepting activity of lutropin, the β‐subunit was devoid of accepting activity. The maximum amount of esterification occurred between 15 and 30 min (at 37° C) depending on the methyl acceptor molecule.

The rate of the methyl esterification of adrenocorticotropin fragments was also studied. While α7–38‐ACTH had less than half of α1–39‐ACTH methyl accepting capacity, α1–17‐ACTH did not serve as methyl acceptor. However, when a mixture of the two fragments was preincubated, the resulting mixture had full α1–39‐ACTH activity.
IMPROVED PROCEDURE OF PURIFICATION OF INORGANIC PYROPHOSPHATASE FROM BACILLUS STEAROTHERMOPHILUS
Authors: SCHREIER E. , SAMEI M. BEKHEIT ABD EL
Published: August 2010

An improved method for isolation of inorganic pyrophosphatase (EC 3.6.1.1.) from Bacillus stearothermophilus is described. The enzyme was purified to more than 90% after two chromatographic steps. A molecular weight of 140 000 daltons was estimated by density gradient centrifugation. The isoelectric point was found to be 4.0.
SYNTHESIS AND SOME PHARMACOLOGICAL PROPERTIES OF [Glu(OMe)4]OXYTOCIN AND [Mpr1, Glu(OMe)4]OXYTOCIN*
Authors: PHOTAKI IPHIGENIA , TZOUGRAKI CHRYSA , KOTSIRA‐ENGONOPOULOS CHRISTINE
Published: August 2010

[Glu(OMe)4] oxytocin (XVI) and [Mpr1, Glu(OMe)4] oxytocin (XVII) bearing a methyl ester group in place of the carboxamide group in position 4 of oxytocin were synthesized by (3 + 6) segment condensation using the S‐trityl group for the protection of the cysteine side chains. Analogue XVI exhibited 10.5 U/mg in vitro uterotonic, and 42 U/mg avian vasodepressor, activity, and analogue XVII 21.4 U/mg and 82 U/mg of the respective activities. Both compounds showed no response in the rat pressor assay.
A FRACTIONAL CHARGE MODEL FOR EMPIRICAL CALCULATIONS OF PEPTIDE‐WATER INTERACTIONS
Authors: VOVELLE FRANÇOISE , MARIUSTTAK
Published: August 2010

The properties of an empirical model of interaction between a water molecule and polar groups of peptides or small peptides are explored. The H2O molecule is represented by a four‐point charges distribution. In electron donor groups, a point charge is located on the axis of the lone pairs orbitals in order to introduce some directionality in hydrogen bonds. The effective potential is approximated by the sum of the coulombic interactions between point charges distribution and of a 6–12 atom‐atom potential. The coefficients of this last potential are first adjusted by simulating the geometry of the water dimer. Equilibrium configurations of associated polar molecules and H2O predicted by the model are found to be in good agreement with those resulting from more sophisticated ab initio SCF calculations. Interactions between H2O and the side‐chains of the cyclic dipeptide C(l‐Thr‐l‐His) are then calculated. It is shown that internal bridging by water is an essential effect of the solvent. The experimental position of the H2O molecule is reproduced, stability of which depends also on intermolecular interactions.
SIMPLE MODIFIED PROCEDURE FOR ENZYMIC DETERMINATION OF RACEMISATION OF HISTIDINE RESIDUES IN PEPTIDE SYNTHESIS
Authors: JONES JOHN H. , RAMAGE WILLIAM I.
Published: August 2010

Details are given of a modified l‐amino acid oxidase procedure for the estimation of racemisation of histidine residues in peptide synthesis.
CHARACTERIZATION OF TURKEY MYELIN BASIC PROTEIN ISOLATED BY A SIMPLE PROCEDURE
Authors: SWANN RAYMOND W. , LI CHOH HAO
Published: August 2010

Highly purified basic proteins have been isolated from bovine and turkey brains by a novel method employing acid‐acetone extraction. The final product gave a single band on polyacrylamide gel electrophoresis at pH 4.3 and in the presence of sodium dodecyl sulfate. Both proteins have arginine at the COOH‐terminus while the NH2‐terminal residue cannot be detected and is probably blocked. A higher ratio of histidine to lysine and a greater proportion of serine and valine was found for the turkey compared with the bovine protein. Both proteins contain one tryptophan and two methionine residues. However, it was found from cyanogen bromide treatment that there is a marked difference in the location of one of the methionine residues, while the tryptophan‐containing peptides liberated after trypsin digestion have different mobilities on peptide maps. When dissolved in water these proteins give a typical random coil curve from circular dichroism (CD)*, whereas in 80% methyl alcohol they assume a 25% α‐helix.
CIRCULAR DICHROISM STUDIES OF SUBSTANCE P AND ITS C‐TERMINAL SEQUENCES
Authors: MEHLIS BURKHARD , RUEGER MARGITTA , BECKER MANFRED , BIENERT MICHAEL , NIEDRICH HARTMUT , OEHME PETER
Published: August 2010

CD spectra of substance P (SP) and its C‐terminal partial sequences have been measured in diluted aqueous solution including variation of hydrogen ion concentration. In the far u.v. region there is an overlapping of the amide CD absorption by the CD of the phenylalanine residue aromatic side chains (217–220 nm). This complex CD absorption is reduced during changes from acidic to alkaline pH, especially in those cases where a phenylalanine residue is at the N‐terminus of the peptide chain. These CD changes dependent on pH are due more to charge effects on the aromatic chromophores than to substantial conformational changes. However, solvation effects on the conformational features of SP peptides caused by the deprotonation of the amino groups have to be taken into account. CD and potentiometric titrations indicate that the N‐terminal α‐amino groups of the SP peptides in general are freely accessible to the solvent. Our studies did not give any evidence of the occurrence of ordered structures of SP peptides in diluted aqueous solution.
CARBON‐13‐N.M.R.‐SPECTRA OF ANTAMANIDE, SEVERAL ANALOGUES AND THEIR ALKALI ION COMPLEXES
Authors: POOK KARL‐HEINZ , NASSAL MICHAEL , BIRR CHRISTIAN , WIELAND THEODOR
Published: August 2010

Natural abundance carbon‐13 Fourier transform n.m.r.‐spectra were obtained of the cyclic decapeptides [Phe4 Val6] antamanide (J); antamanide (II); [Tyr5] antamanide (III); [Ala1] antamanide (IV) and their ion complexes (I)‐Na+, (II)‐Na+, (II)‐Li+, (III)‐Na+ and (IV)‐Na+, Based upon literature data, systematic comparisons and model compounds, a line assignment approach was performed for the majority of the aliphatic carbons. The spectra of the [Ala Val6] analogues (V) (Bystrov et al., 1972) and II (measured in CD3CN Patel, 1973a, b) and their complexes were also assigned. Characteristic chemical shift variations observed upon complex formation were calculated (free peptide/Me+ complex) for a number of corresponding carbons, revealing shift changes up to 2.4 p.p.m. Preliminary calculations of the angle at selected prolines for some ion complexes are included.
ISOLATION AND CHARACTERIZATION OF THREE LUTROPIN ISOHORMONES FROM THE PITUITARY GLAND OF THE OSTRICH (STRUTHIO CAMELUS)
Authors: OOSTHUIZEN M. M. J. , OELOFSEN W. , SCHABORT J. C. , NEITZ A. W. H. , VILJOEN C. C.
Published: August 2010

Avian lutropin has been isolated from pituitary glands of the ostrich (Struthio camelus) in three homogeneous forms (designated isohormones). The different homogeneous forms of ostrich lutropin were characterized physically and chemically in terms of molecular weight, electrophoretic mobility, isoelectric points, amino acid and carbohydrate composition. From these characteristics it was evident that the isohormones are very similar. The differences between these isohormones can be attributed to differences in carbohydrate composition, especially sialic acid.
TRYPTOPHAN REPLACEMENT IN THE C‐TERMINAL OCTAPEPTIDE OF CCK‐PZ
Authors: RAJH HUBERTUS M. , MARIMAN EDWIN C.M. , TESSER GODEFRIDUS I. , NIVARD RUTGER J.F.
Published: August 2010

Six sulphated octapeptide amide analogues, representing the C‐terminal sequence of pancreozymin have been prepared in addition to the natural sequence. The tryptophyl residue which is essential for biological activity was replaced in this series by structurally, closely related analogues each of which resemble the properties of the original moiety to varying extents. The syntheses were so conducted that the C‐terminal tetrapeptide amides (modified tetragastrins) could be secured. The results of the measurement of one of the biological parameters are indicated. The activity was determined to elucidate which of the physicochemical properties of the tryptophyl residue is most essential for exertion of biological activity in the described test.
EFFECT OF SODIUM DODECYL SULFATE, ACID, ALKALI, UREA AND GUANIDINE HYDROCHLORIDE ON THE CIRCULAR DICHROISM OF α‐GLOBULIN OF SESAMUM INDICUM L
Authors: PRAKASH V. , NANDI R. K. , JIRGENSONS B.
Published: August 2010

The circular dichroic spectra of α‐globulin have been obtained under various solution conditions of sodium dodecyl sulfate, acid, alkali, urea and guanidine hydrochloride. The protein in phosphate buffer pH 7.4, 0.2 M has about 25%β‐structure and 5%α‐helix, the rest being aperiodic or irregular structure. Sodium dodecyl sulfate induced more α‐helical structure in the protein. The protein had nearly 20%α‐helix at 1 times 10‐2 M SDS. At extreme acid or alkaline pH, the protein had no α‐helix with β‐structure decreasing with further extremes of pH. The protein is represented by 100% aperiodic structure in 6.6 M urea and in 6.0 M guanidine hydrochloride solutions. The above results are discussed in view of some of the earlier results with regard to the association‐dissociation and denaturation behavior of α‐globulin under various solution conditions.
SYNTHESIS OF HUMAN β‐ENDORPHIN IN SOLUTION USING BENZYL‐TYPE SIDE CHAIN PROTECTIVE GROUPS
Authors: TZOUGRAKI CHRYSA , MAKOFSKE RAYMOND C. , GABRIEL THOMAS F. , MICHALEWSKY JOSEPH , MEIENHOFER JOHANNES , LI CHOH HAO
Published: August 2010

A solution synthesis of human β‐endorphin (β‐EP) was carried out by condensation of protected peptide segments bearing Nα‐tert.‐butyloxycarbonyl groups and benzyl‐derived groups for the protection of functionalities in amino acid side chains. Five intermediate segments were assembled in a stepwise manner starting at the carboxyl terminus. Thus, the segment of sequence region (27–31) was coupled to segment (22–26) by the azide method. Segment (19–21) was incorporated into the growing chain by azide coupling, and segment (10–18) by dicyclohexylcarbodiimide coupling in the presence of 1‐hydroxybenzotriazole (DDC‐HOBt). Solubility problems in condensing the ensuing 22‐peptide with segment (1–9) by DDC‐HOBt were overcome by using a dimethylformamidephenol mixture as a solvent. Protecting group cleavage by Na in liquid NH3 was much superior to liquid HF which gave rise to many decomposition products. Homogeneous βn‐EP indistinguishable from authentic material in physiochemical and biological properties, was obtained in a single preparative reversed phase liquid chromatographic step after protecting group cleavage.
STUDIES ON THE SYNTHESIS OF PROTEINASE INHIBITORS
Authors: TERADA SHIGEYUKI , SATO KAZUKI , KATO TETSUO , IZUMIYA NOBUO
Published: August 2010

Several heterodetic cyclic nonapeptides modeled after the disulfide loops of Bowman‐Birk inhibitor which involve either the anti‐tryptic or anti‐chymotryptic region have been synthesized by the conventional method. The inhibitory properties and the stabilities of these peptides toward trypsin and chymotrypsin, as well as the properties of dimers of the above nonapeptides, were examined.
NUCLEAR MAGNETIC RESONANCE STUDY OF A HYBRID OF BOVINE AND RAT RIBONUCLEASE
Authors: BEINTEMA JAAP J. , LENSTRA JOHANNES A.
Published: August 2010

A hybrid RNase S', consisting of synthetic S‐peptide of rat ribonuclease and bovine S‐protein, was studied by 1H n.m.r. to determine the effects of the many N‐terminal amino acid replacements in rat S‐peptide as compared to bovine S‐peptide. As judged from the aromatic resonances, the conformation of the hybrid RNase S' is essentially identical to the conformation of bovine RNase S'. Notably, the active‐site histidines 12 and 119 were not affected by the substitutions in the S‐peptide, confirming earlier findings that the catalytic properties of naturally occurring RNases are modulated by the S‐protein part of the molecule only. However, the resonances of Tyr‐25 and His‐48, which in RNase A are involved in a pH‐dependent conformational transition, appeared to be different in the hybrid RNase, demonstrating that amino acid replacements may influence the structure of the protein locally.
IMMUNOCHEMICAL STUDIES OF ADRENOCORTICOTROPIN USING TRITIUM LABELED HORMONE
Authors: RAO A. JAGANNADHA , BEHRENS CATHERINE , RAMACHANDRAN J.
Published: August 2010

A radioimmunoassay using specifically tritiated corticotropin (3H‐ACTH) of high specific radioactivity (46 Ci/mmol) and full biological activity has been developed. The assay is sensitive to ACTH in the range 200 pg‐10 ng. 50% displacement of 3H‐ACTH is produced by less than a twofold excess of unlabeled hormone. Immunochemical studies with various ACTH analogs showed that the antiserum recognized the intact molecule better than any of the fragments.
HYDRODYNAMIC AND QUASI‐ELASTIC LIGHT SCATTERING STUDIES ON THE 12S GLOBULIN FROM RAPESEED*
Authors: SCHWENKE KLAUS DIETER , SCHULTZ MANFRED , LINOW KARL‐JOACHIM , GAST KLAUS , ZIRWER DIETRICH
Published: August 2010

The 12S globulin, one of the major storage proteins of rapeseeds, has the following physico‐chemical constants, as determined by ultracentrifugation, quasi‐elastic light scattering measurements and gel chromatography: sedimentation coefficient s020, w= 12.7 times 10‐13 s; diffusion coefficient (quasi‐elastic light scattering) D020, w= 3.8 × 10‐7 cm2 s‐1; Stokes radius (by quasi‐elastic light scattering) Rs= 5.7 nm and (by gel chromatography) Rs= 5.5 nm; partial specific volume (calculated from the amino acid composition) v̄= 0.729 mlg‐1; molecular weight Ms, D = 300, 000 daltons, Ms, Rs= 294,000 daltons (Rs from the gel chromatography); frictional ratio f/fo = 1.28.
STUDIES ON PEPTIDES
Authors: YAJIMA HARUAKI , TAKEYAMA MASAHARU , KOYAMA KANAME , TOBE TAKAYOSHI , INOUE KAZUTOMO , KAWANO TAMOTSU , ADACHI HIDEKI
Published: August 2010

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken VIP was synthesized in a conventional manner, using a new arginine derivative, NG ‐mesitylene‐2‐sulfonylarginine, Arg(Mts). Treatment of a fragment, Z(OMe)‐Thr‐Asp‐Asn‐Tvr‐NHNH2 with methanesulfonic acid or HBr was found to give a product with a low recovery of Asp, after aminopeptidase digestion. Ring closure of the Asp‐Asn unit seemed to be responsible for this phenomenon. Deprotection with HF or TFA exhibited definitely less such a tendency. In the final step of the synthesis, all protecting groups, including the Mts group, were removed by HF in the presence of m‐cresol and the deprotected peptide was purified by ion‐exchange chromatography on CM‐cellulose followed by isoelectric focusing in Ampholine pH 9–11. Synthetic peptide exhibited the identical Rf value with that of natural chicken VIP and was active as the natural peptide.
STUDIES ON PEPTIDES
Authors: YAJIMA HARUAKI , FUJII NOBUTAKA , HIROTA YORIKO , NASADA YUKO , HIRAI YUKO , NAKAJIMA TERUMI
Published: August 2010

The tetradecapeptide amide corresponding to the entire amino acid sequence of a wasp venom (polistes mastoparan), isolated from Polistes jadwagae, was synthesized using the trifluoroacetic acid‐thioanisole deprotecting procedure.
PHYLOGENY OF NEUROHYPOPHYSEAL HORMONES. Vasopressin Polymorphism in Three Kangaroo Species
Authors: CHAUVET MARIE‐THERESE , HURPET DOMINIQUE , CHAUVET JACQUELINE , ACHER ROGER
Published: August 2010

The neurohypophyseal hormones of three species of Australian marsupials belonging to the family Macropodidae (Macropus rufus, Macropus giganteus, Macropus robustus). have been investigated. In these species three active peptides have been disclosed, one with oxytocic activity and two with pressor activity. The three hormones are present in all the individual glands which have been examined. Purification has been undertaken by ion‐exchange chromatography and paper chromatoelectrophoresis. The more abundant of the pressor hormones (about 80% of the total pressor activity) has been identified as lysine‐vasopressin by its biological properties, its chromatographic behaviour and its amino acid composition. The second pressor principle, more basic, has not yet been chemically characterized but its pharmacological properties and its chromatographic behaviour suggest that it is closely related or identical to arginine‐vasopressin. Dimorphism of vasopressins seems peculiar to Metatheria since it has not been observed in Protheria and very rarely in Eutheria.
ISOLATION, CHARACTERIZATION AND AMINO ACID SEQUENCE OF β‐LIPOTROPIN FROM HUMAN PITUITARY GLANDS
Authors: LI CHOH HAO , CHUNG DAVID
Published: August 2010

β‐Lipotropin has been purified to homogeneity from human pituitary glands. It consists of 89 amino acids with a molecular weight of 11 700. The lipolytic potency of the human hormone is identical to that of ovine β‐LPH. The amino acid sequence has been determined as follows: H‐1Glu‐Leu‐Thr‐Gly‐Gln‐Arg‐Leu‐Arg‐Gln‐10Gly ‐ Asp‐Gly‐Pro ‐ Asn ‐Ala‐Gly‐Ala‐Asn ‐ Asp‐20Gly‐Glu‐Gly‐Pro‐Ala‐Ala‐Asp ‐ Leu‐Glu‐His‐30Ser‐Leu ‐ Leu‐Val‐ Ala‐Ala‐Glu‐Lys‐Lys‐Asp‐40Glu‐Gly‐Pro‐Tyr‐Arg ‐ Met‐Glu‐His‐Phe ‐ Arg‐50Trp‐Gly‐Ser‐Pro‐Pro‐Lys‐Asp‐Lys‐Arg‐Tyr‐60Gly‐Gly‐Phe‐Met‐Thr‐Ser‐Glu‐Lys‐Ser‐Gln‐70Thr‐Pro‐Leu‐Val‐Thr‐Leu‐Phe‐Lys‐Asn‐Ala‐80Ile‐Ile‐Lys‐Asn‐Ala‐Tyr‐Lys‐Lys‐Gly‐89Glu‐OH. When compared with the structure of the ovine hormone, the sequence at the NH2 ‐terminal 33 residues exhibits considerable variability.
DISTANCE GEOMETRY ANALYSIS OF THE N.M.R. EVIDENCE ON THE SOLUTION CONFORMATION OF BLEOMYCIN
Authors: CRIPPEN GORDON M. , OPPENHEIMER NORMAN J. , CONNOLLY MICHAEL L.
Published: August 2010

Conformational constraints derived from n.m.r. experiments, X‐ray data and the known stereochemistry have been used to investigate by the distance geometry method the range of allowed solution conformations for Cu(II): P‐3A (a biosynthetic precursor of bleomycin), Fe(II):bleomycin:carbon monoxide, and Zn(II): bleomycin. The experimental data have been found to be self‐consistent and lead to the following observations. 1) Designation of the ligands and the dihedral angles available from vicinal coupling constants are not sufficient to define uniquely the geometry around the metal. 2) When only five bleomycin ligands are invoked (e.g. Cu(II):P‐3A or Fe(II):bleomycin:carbon monoxide) there is considerable freedom in the allowed coordination scheme around the metal, but some regions of the molecule have well determined conformation. 3) Introduction of a sixth bleomycin ligand, as in Zn(II): bleomycin, considerably constrains the conformational freedom of the groups coordinated to the zinc. The utility of the distance geometry approach for analysis of data and design of experiments is discussed.
FLUORESCENCE SPECTRA OF LYSOZYME EXCITED AT 305 NM IN PRESENCE OF UREA
Authors: RAO M.V.R. , ATREYI M. , RAJESWARI M.R.
Published: August 2010

Tryptophan 108 of hen egg white lysozyme was selectively excited at 305 nm and fluorescence spectra were recorded as a function of pH (2–9) and concentration of urea (0–8 M). Urea at low concentrations (1–4 M) quenches markedly the Trp 108 fluorescence around pH 7; the Λmax, however, remains unaltered. The fluorescence quenching by urea is most likely due to local conformational changes around Trp 108 in the active site region of the enzyme. Substantial unfolding of the enzyme, however, was brought about by 4M urea below pH 3, and by 7 M urea at pH 10.3, as indicated by a marked red shift in the Λmax of the fluorescence emission.
β‐STRUCTURE OF POLYPEPTIDES IN NON‐AQUEOUS SOLUTIONS: II. Side‐Chain Orientation
Authors: RAGHAVENDRA K. , ANANTHANARAYANAN V.S.
Published: August 2010

The specific side‐chain orientations of the phenyl group in the polypeptides poly‐S‐benzyl‐l‐cysteine, poly‐S‐carbobenzoxy‐l‐cysteine and poly‐O‐carbobenzoxy‐l‐serine in the β‐structure have been studied by spectral measurements in solution. All the three polypeptides exhibit aromatic CD bands, indicating the asymmetric placement of the side‐chain phenyl rings when the polypeptide backbone takes up the antiparallel β‐structure. Supporting evidence for this is derived from n.m.r. spectra of the polypeptides, which show up field shift of the phenyl protons due to the stacking of the aromatic rings. Molecular model building studies reveal the stacking of alternate phenyl groups along the polypeptide chain.
FIELD DESORPTION MASS SPECTROMETRY
Authors: DEUTSCH JOSEPH , GILON CHAIM , CHOREV MICHAEL
Published: August 2010

In this article we present preliminary results of the application of potassium cationized field desorption mass spectrometry as an additional technique for the elucidation of structure and evaluation of purity of oligopeptides such as C‐terminal penta‐ and hexapeptide analogs of substance P. In the resultant mass spectra both a protonated and a cationized molecular ion, MH+ and MK+respectively, were observed. The m/z values of the two peaks were in agreement with the calculated molecular weights. The ratio between the relative abundancies of these ions (MH+/MK+) was found to be characteristic of the particular peptide and thus useful in the assessment of their purity. Among the 13 peptides studied, only two gave pyroly tic fragmentation leading to a more complex spectra.
Conformational energy calculations on Substance P
Authors: MANAVALAN P. , MOMANY F.A.
Published: August 2010

The conformational and spacial configurations of the biologically active undecapeptide, Substance P, were studied using conformational energy calculations. Low energy conformers of residues 1–5 and 6–11 were found by energy minimization and the two fragments were then combined to find low‐energy structures for Substance P. Several configurational classes were found with different backbone conformations. A comparison of the final low‐energy structures with data from biological tests on analogs of Substance P gives some insight into the conformation required for interaction at the biological receptor.
Reduction of methionine sulfoxide in peptide synthesis by use of 2‐mercaptopyridine in liquid hydrogen fluoride
Authors: YAMASHIRO DONALD
Published: August 2010

Methionine d‐sulfoxide can be reduced to methionine in liquid hydrogen fluoride in the presence of 2‐mercaptopyridine. The utility of the reaction in peptide synthesis was demonstrated by preparation of the model peptide H‐Leu‐Gly‐Arg‐Leu‐Gly‐Met‐Phe‐OH (I) by the solid‐phase method. When the peptide resin H‐Leu‐Gly‐Arg(Tos)‐Leu‐Gly‐Met(d‐sulfoxide)‐Phe‐resin is treated in liquid HF in the presence of the standard scavenger anisole, H‐Leu‐Gly‐Arg‐Leu‐Gly‐Met(d‐sulfoxide)‐Phe‐OH (II) is obtained in ca. 66% overall yield with no detectable trace of I. When the same peptide resin is treated in like manner in the added presence of 2‐mercaptopyridine, I is obtained in ca. 63% overall yield with no detectable trace of II.
Application of pattern recognition techniques to the analysis of protein crystal structure data. I. Characteristics of per‐residue side chain contact frequency distributions
Authors: BOHACEK REGINE S. , DAVISON BRUCE , ROBERTS DAVID C.
Published: August 2010

A statistical study of amino acid side chain contact interactions was carried out using a data set based on 36 protein structures. For each type of amino acid, a distribution of per‐residue inter‐side‐chain contacts was obtained, over the observed span of zero to 11 contacts per residue. Significant observations included the following: 1) The mean number of inter‐side‐chain contacts is proportional to side chain surface area with the exception of Lys and Arg. 2) The mean number of contacts was greater for amino acids in β‐sheet relative to α‐helical regions. 3) The more polar or surface‐loving amino acids exhibited non‐normal distributions, whereas distributions for the non‐polar or interior‐loving amino acids fell within accepted limits of normality.
Specificity of spatial clustering of amino acid residues as an evolutionary trend in proteins
Authors: VISWANADHAN V.N. , SUNDARAM K.
Published: August 2010

Three‐dimensional structures of cytochrome c and myoglobin molecules together with amino acid substitution data on these proteins from a close range of taxa highlight an interesting evolutionary trend. Assuming three‐dimensional structures of homologous proteins to be largely similar, the distribution of amino acid residue types around one another was studied. The application of information theory to three dimensional arrangement of amino acid residues revealed that in general, the higher the taxonomic order, the more specific is the clustering of residue types with one another.
d· Ala5 analog of the elastin polypentapeptide. Physical characterization
Authors: URRY D.W. , TRAPANE T.L. , WOOD S.A. , WALKER J.T. , HARRIS R.D. , PRASAD K.U.
Published: August 2010

The d ° Ala5 analog, (l · Val1‐l · Pro2‐Gly3‐l · Val4‐d · Ala5)n, of the polypentapeptide (PPP) of elastin is synthesized and characterized by a series of physical methods. Carbon‐13 and proton nuclear magnetic resonance spectroscopies are used to verify purity and, by means of solvent dependence of peptide C ‐O chemical shift and of temperature dependence of peptide NH chemical shift, to establish by comparison with the PPP of elastin the presence and increased stability of the Type II Pro2‐Gly3 β‐turn. The temperature dependence of aggregation in water to form a viscoelastic phase called the coacervate is reported for several concentrations. Comparison of carbon‐13 nuclear magnetic resonance spectra obtained under identical conditions for the coacervate states of the PPP of elastin and the d · Ala5 analog shows the effect of replacing the Gly5 residue by a d · Alas residue to be one of greatly restricting mobility of the polypeptide chain. Scanning electron micrographs, of the coacervate alone and of the coacervate cross‐linked and compounded to a Dacron fabric before and after stress‐strain studies, are reported which show the d · Ala5 PPP matrix to rupture during the stresses of drying and of stretching while wet. Thus, the effect of adding a methyl moiety to the Gly5 residue of the PPP of elastin is to decrease markedly the mobility of the polypeptide chain and to destroy elasticity. The results are presented as a test of the proposed librational entropy mechanism of elasticity of the PPP of elastin.
Spectroscopic study on the structure and stability of beef liver arginase
Authors: ROSSI VALDANO , GRANDI CLAUDIO , DALZOPPO DANIELE , FONTANA ANGELO
Published: August 2010

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far‐ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrsoine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the trytophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+‐ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far‐ultraviolet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77° was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+‐ions for 10 min; in the presence of EDTA, Tm is shifted to 55°. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+‐ion binding.
Iodogen‐catalyzed iodination of transferrin
Authors: REGOECZI ERWIN
Published: August 2010

Transferrin (human, rabbit) labels at low efficiency (1%‐10%) with 125I when reaction of 0.5–0.7 ng of I‐ (8–10μCi) with 20μg of the protein is catalyzed by iodogen in a constant volume of 0.1 ml. Microiodination by this technique was therefore analyzed with regard to the relative proportions of the reactants, oxidant requirement, and timing. In vials giving a reaction volume‐to‐active surface ratio of 0.88, efficiency was independent of the amount of iodogen in the range from 1 μg to 15 μg, and prolongation of the reaction beyond 1 min failed to improve yields. In contrast, the amount of I‐ present was decisive. Butanol/NH4OH chromatograms of iodination reactions carried out with 0.6 ng or 20 ng of I‐ showed 3–4 radioactivity peaks, the relative proportions of which markedly depended on the amount of I‐ present originally. A link was established between labeling efficiency and chromatographic profile of the I‐ derivatives formed during oxidation. Dual‐label experiments in rats showed that transferrin (20μg) can be labeled using iodogen (1–5μg, 1 min) to behave indistinguishably from its IC1‐labeled counterpart. However, prolonged exposure to more oxidant progressively damaged the protein. The damage was independent of substituting I and it manifested itself in increased protein binding to the anion exchange resin, Dowex 1‐X8. Over 99.5% of the labeled residues in iodotransferrin were mono‐ and diiodotyrosines (MIT, DIT). DIT content of the protein increased linearly with the number of I atoms substituted. At comparable levels of substitution, more label was present as MIT after using iodogen than after using ICl. Electrophoretic data are presented regarding homogeneity of the label as obtained after iodinating transferrin by different methods and to varying extents.
Synthesis of phosphonic analogs of enkephalins
Authors: Kupczyk‐Subotkowska Lidia , Mastalerz Przemyslaw
Published: August 2010

Eight analogs of enkephalins containing the phosphonic analogs of Gly, Leu, Met, Phe and Pro have been synthesized by standard procedures in solution. The key intermediates in the synthesis were pure diastereomers of dialkyl N‐L‐phenylalanyl/aminoalkanephosphonates obtained from racemic dialkyl aminoalkanephosphonates by coupling with phenylalanine and resolution of the resulting mixture of L, D and L, L dipeptides by means of column chromatography.
Analogs of phalloidin
Authors: WIELAND THEODOR , MIURA TAMIKO , SEELIGER ANNEMARIE
Published: August 2010

The analogs [D‐Abu2‐Lys7]‐phalloin (1) and [D‐Ala2‐Leu7]‐phalloin (2) are obtained by cyclization of the monocyclic thioether peptides 5 and 7 with DCCI and diphenylphosphoro‐azidate respectively. For the synthesis of 5 and 7 an intramolecular Savige‐Fontana reaction of the linear heptapeptide tert.‐butylesters 4 and 6 is applied. On treatment in dilute solution with TFA the classically synthesized peptides 4 and 6 lose their N‐terminal Boc groups thus giving rise to the reaction of the Hpi residues with the released cysteine SH‐groups. The lysine analog 1 binds to F‐actin with an association constant of 1.3 times 10‐6M whereas analog 2 exhibits practically no affinity. By reaction of 1 with tetramethyl‐rhodaminyl‐isothiocyanate a fluorescent derivative, rhoda‐minyl‐lysine‐phallotoxin (RLP), is obtained as a novel fluorescent probe for the visualization of F‐actin in cell preparations.
Partially modified retro‐inverso peptides
Authors: CHOREV MICHAEL , GOODMAN MURRAY
Published: August 2010

Several synthetic routes are reported to prepare the hetero diprotected 1,1‐diaminoalkanes from N‐acylated amino acids or peptides for incorporation into partially modified retro‐inverso peptides. The Curtius rearrangement was carried out on the N‐protected aminoacyl azides obtained from the N‐protected aminoacyl hydrazide by nitrosyl chloride or by sodium azide reaction with an appropriate mixed carboxylic carbonic acid anhydride. The resulting isocyanate was allowed to react with alcohol to give a urethane‐type protecting group or, via a “one‐pot” approach, directly with a carboxyl carrying component to yield the modified (reversed) peptide bond. The carboxyl component can be either an N‐acylated amino acid or a malonic acid. The more standard route involves selective deprotection of the 1,1‐diaminoalkane residue followed immediately by coupling with a carboxyl component to yield the same modified peptide derivative.
Chromatin associated protease from calf thymus
Authors: DYSON MICHAEL , WALKER JOHN. M.
Published: August 2010

We have identified and purified a chromatin bound protease from calf thymus. The purified enzyme cleaves histone H1 when whole histone is used as substrate. The enzyme is inhibited by Soya bean trypsin inhibitor, but not by PMSF, EDTA, pepstatin or Ellman's reagent. Further studies have shown that H1d̀ and histone H5 are also cleaved by this enzyme, and that when HMG 1 and 2 are used as substrate HMG1 alone is degraded to give specific degradation products. We have also shown that the enzyme co‐isolates with whole histone prepared by acid extraction of calf thymus nuclei, but interestingly it is not present in whole histone samples prepared in the same manner from pig thymus. A second chromatin bound protease which specifically cleaves histone H3 when whole histone is used as substrate has also been identified during the course of this work.
Backbone side chain interactions in peptides.
Authors: Marraud M. , Aubry A.
Published: August 2010

The high content of serine in β‐folded regions of proteins may be the consequence of some specific interaction between the peptide backbone and the hydroxyl group of the Ser side‐chain. The resolution of the X‐ray structures of three peptides with the Pro‐Ser sequence protected on both ends by amide and/or ester functions indicates that the Ser NH bond is a proton donating group to the Ser Oχ atom in the solid state. The present study deals with spectroscopic investigations on five Ser‐containing model dipeptides with the l‐Pro‐d‐Ser 1, l‐Pro‐l‐Ser 2, l‐Ala‐l‐Ser 3, l‐Ser‐l‐Ala 4 and l‐Ser‐Gly 5 sequences protected on their N and C‐termini by tBuCO and NHMe groups, respectively. The N–H.Oγ interaction found in the solid state of 1 and 2 is at least partly retained in solution and its occurrence in X‐Ser sequences is fully compatible with β‐folding. The same is not true for Ser‐X sequences in which the competition between the typical β‐turn i + 3 rarr; i hydrogen bond and the N–H.Oγ interaction results in lower contents of β‐folded conformers. Because of this latter interaction, the rotamer III (χ1 60°) is the most frequent disposition of the Ser Cα–Cβ bond in all five derivatives.
Monte Carlo study on local and small‐amplitude conformational fluctuation in hen egg white lysozyme
Authors: Wakana Hiromitsu , Wako Hiroshi , Saito Nobuhiko
Published: August 2010

Local and small‐amplitude conformational fluctuations in hen egg white lysozyme around its native conformation were studied by the Monte Carlo simulation with conformational energy calculation. In order to carry out such a simulation in a shorter computation time, the following method was devised: at each step of the simulation a segment of consecutive four residues, say, i to i + 3, is chosen at random from N residues and then the small conformational change of the segment is performed so that the conformations of the two blocks of residues 1 to i — 1 and i + 4 to N as well as the mutual location of the two blocks are not changed. In this simulation it was found that calculated atomic displacements and fluctuations of dihedral angles well reflect the characteristics of local conformations, for example, stiffness of regular secondary structures and flexibility of non‐regular structures, especially of the regions around the lips of the active‐site cleft and of the region that undergoes conformational change on ligand binding to the active site. The flexibility of these regions is probably necessary for the reaction of the active site to the ligand. A close correlation between the solvent accessibility of the side chain of each residue and its flexibility was also observed. Furthermore, it was shown that the results obtained in this study are in a good agreement with the same properties observed in analyzing temperature factors derived from refinement of X‐ray data of the protein.
Conformational study of glycopeptides
Authors: ISHII HIROSHI , INOUE YOSHIO , CHÛJÔ RIICHIRÔ
Published: August 2010

The conformational feature has been studied by n.m.r. spectroscopy on the compounds, Boc‐Asn‐NHMe, Boc‐Asn‐Gly‐NHMe, Boc‐Gly‐Asn‐NHMe, and their glycosylated derivatives. From the temperature dependence of the amide proton chemical shifts and vicinal coupling constants, little change was confirmed in the peptide conformation upon N‐glycosylation. There is no particular intramolecular interaction between the peptide and carbohydrate moieties. Boc‐Asn‐Gly‐NHMe takes, to some extent, a folded structure with a hydrogen bond involving the amide proton of N‐methylamide group. This backbone conformation is also preferable in the corresponding glycopeptide.
Vibrational analysis of peptides, polypeptides, and proteins
Authors: BANDEKAR JAGDEESH , KRIMM S.
Published: August 2010

The normal modes have been calculated for three kinds of low energy γ‐turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a γ‐turn, a mirror‐related γ‐turn, and an inverse γ‐turn of CH3‐CO‐(L‐Ala)n‐NH‐CH3, with n = 3 and n = 5, and for certain 14C and 15N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and γ‐turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.
Influence of deamidation(s) in the 67–74 region of ribonuclease on its refolding
Authors: VENKATESH YELDUR P. , VITHAYATHIL PAUL J.
Published: August 2010

The influence of chemical mutation featuring the selective conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, on the kinetics of refolding of reduced RNase has been studied. The monodeamidated derivatives of RNase A, viz. RNase Aa1a, Aa1b, and Aa1c having their deamidations in the region 67–74, were found to regain nearly their original enzymatic activity. However, a marked difference in the kinetics of refolding is seen, the order of regain of enzymic activity being RNase A > Aa1c Aa1a > Aa1b. The similarities in the distinct elution positions on Amberlite XE‐64, gel electrophoretic mobilities, and u.v. spectra of reoxidized and native derivatives indicated that the native structures are formed. The slower rate of reappearance of enzymic activity in the case of the monodeamidated derivatives appears to result from altered interactions in the early stages of refolding. The roles of some amino acid residues of the 67–74 region in the pathway of refolding of RNase A are discussed.
An algorithm for reconstructing protein sequences
Authors: PETRILLI P.
Published: August 2010

A computer program was designed to attempt the reconstruction of a protein primary structure when sequence data for a single set of peptides and amino acid compositions for other set(s) are available.
Presence of an amphipathic helical segment and its relationship to biological potency of calcitonin analogs
Authors: EPAND RICHARD M. , EPAND RAQUEL F. , ORLOWSKI RONALD C.
Published: August 2010

The conformational properties of a number of calcitonin analogs were studied by circular dichroism. The ability of dimyristoylphosphatidylglycerol, lyso‐phosphatidycholine or sodium dodecyl sulfate to induce the formation of more highly ordered structures in these peptides was also assessed by circular dichroism. In all cases sodium dodecyl sulfate induced the largest change in the circular dichroism spectra of the peptides. Salmon calcitonin and its analogs were slightly more helical in the presence of the anionic phospholipid than in the presence of the zwitterionic detergent lysophosphatidylcholine while the reverse is true for human calcitonin and its analogs. Some of the calcitonin analogs convert turbid suspensions of phosphatidylglycerol to a clear solution from which the phospholipid is no longer readily sedimentable by centrifugation. Several of the physical properties of these peptides could be correlated with their biological activity. Generally peptides which showed no hypo‐calcemic activity had the least negative mean residue ellipticities at 222 nm. Only biologically active analogs were able quantitatively to solubilize dimyristoyl‐phosphatidylglycerol and in this solubilized form the peptides have a higher helical content. More active derivatives exhibit larger increases in helix content in the presence of this phospholipid. Inactive analogs had the least negative mean residue ellipticities at 222 nm in the presence of lysophosphatidylcholine or sodium dodecyl sulfate. Thus, the ability of a calcitonin analog to form structures of higher helical content in the presence of amphiphiles is a requirement for the analog to exhibit high potency in assays of biological activity.