AMINO ACID COMPOSITIONS AND N-TERMINAL AMINO ACID OF ALL THE TRYPTIC PEPTIDES FROM THE α AND β POLYPEPTIDE CHAINS OF ADULT HEMOGLOBIN OF THE JAPANESE MONKEY (Macaca fuscata fuscata)

Globin prepared from adult hemoglobin of the Japanese monkey was separated into α and β polypeptide chains by the countercurrent distribution method. All the tryptic peptides obtained from these polypeptide chains were isolated and purified by column and paper chromatography. The amino acid compositions and the N-terminal amino acid of these tryptic peptides were analyzed. The results thus obtained suggested amino acid exchanges between Japanese monkey and human hemoglobin, that is, at four positions in α polypeptide chain and seven positions in β polypeptide chain. In comparison with the α and β polypeptide chains of adult hemoglobin from the rhesus monkey, the α chain is quite the same, and it is considered that there is one amino acid exchange in the β chains. The outline of this work was presented at Symposium 5 (Biochemical Polymorphisms in Man and Other Primates and their Anthropological Implications) of the 8th International Congress of Anthropological and Ethnological Science (3–10 September 1968, Tokyo     

AMINO ACID COMPOSITIONS OF ALL THE TRYPTIC PEPTIDES FROM THE α AND β POLYPEPTIDE CHAINS OF ADULT HEMOGLOBIN OF THE SLOW LORIS (Nycticebus coucang)

Hemoglobin was prepared from blood of adult animals of slow loris, and furthermore a and β polypeptide chains of the globin were separated by column chromatography. These two kinds of polypeptide chains were respectively digested with trypsin. The tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography, and then each amino acid composition was analyzed.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

THE PURIFICATION OF PEPTIDES BY PARTITION CHROMATOGRAPHY BASED ON A HYDROPHOBICITY SCALE*

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 R_M 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.     

THE PRIMARY STRUCTURES OF α AND β CHAINS OF ADULT HEMOGLOBIN OF THE JAPANESE MONKEY (Macata fuscata fuscata)

Amino acid sequence of the tryptic peptides from the α and β chains of adult hemoglobin of the Japanese monkey were analyzed by partial hydrolyses with enzymes, the DNP method, PTC method and others after the isolation and purification of these peptides. Furthermore, the primary structures of the α and β chains of adult hemoglobin from the Japanese monkey were obtained by homology between these results and those of human adult hemoglobins. These primary structures show a high similarity to the primary structures of the α and β chains of adult hemoglobins from the human and rhesus monkey.     

Isolation of large peptides derived by cyanogen bromide cleavage of thermolysin using fast protein liquid chromatography (FPLC)

The recently introduced fast protein liquid chromatography (FPLC) system of Pharmacia (Uppsala, Sweden) was employed to isolate rather large peptides derived from thermolysin by selective chemical fragmentation at methionine in positions 120 and 205 of the polypeptide chain of 316 amino acid residues. Thermolysin was cleaved under conditions of limited fragmentation in order to produce, besides fragments 1–120, 121–205 and 206–316, the overlapping fragments 1–205 and 121–316. These polypeptides were separated employing prepacked Mono Q or Mono S columns (quaternary ammonium and sulfonic acid support, respectively). The columns were equilibrated with acetate-7 M urea buffer, pH 5.0 or 6.0, and eluted with a gradient of sodium chloride or acetate. Separations were achieved in 10–20 min and were carried out also at a semipreparative level (1–3 mg per run). All five protein fragments were isolated in homogeneous form, as judged by amino acid analysis and electrophoresis. Considering that protein fragmentation with cyanogen bromide is the most commonly used procedure to achieve selective chemical fragmentation of a polypeptide chain, these results indicate that FPLC with ionic exchangers can be usefully employed to isolate rather large protein fragments especially suitable for automatic sequence analysis with the sequenator.     

STUDIES ON PEPTIDES

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken VIP was synthesized in a conventional manner, using a new arginine derivative, N^G -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 THE PRIMARY STRUCTURE OF BISON PANCREATIC RIBONUCLEASE

Bison pancreatic ribonuclease was isolated by affinity chromatography. Thermolysin and tryptic digestion of denaturated protein, and subtilisin digestion of native protein yielded peptides, which were purified and submitted to amino acid analysis. These peptides, together with partial sequence data obtained by Stewart & Stevenson (16) overlap the entire amino acid sequence of bison ribonuclease. No differences with bovine ribonuclease were found, although there may be differences in state of amidation of some residues.     

THE PRIMARY STRUCTURE OF MUSKRAT PANCREATIC RIBONUCLEASE

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32–33) and Ser-Arg (tentative position 75–76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23–25 and 99–102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31–42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44–52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn- Val-Thr (62–64).     

作为乙肝疫苗的新型结构肽的设计与合成

在乙肝病毒表面抗原结构研究的基础上,自行设计了三个新型结构的肽疫苗,采用Boc化学和Fmoc化学相匹配的策略,应用固相肽逐步合成法合成,产物经氨基酸分析鉴定。     

THE AMINO ACID SEQUENCE OF MYOGLOBIN FROM THE MOLLUSC Aplysia limacina

The amino acid sequence of the globin prepared from the myoglobin contained in the radular muscle of the gasteropod mollusc Aplysia limacina was determined. According to the results thus obtained, the polypeptide chain of Aplysia limacina myoglobin is composed of 145 amino acid residues, in good agreement with the overall number of amino acid residues contained in the polypeptide chain of the vertebrate myoglobins studied to date. A particularly interesting feature of the myoglobin studied appears to be the presence in its polypeptide chain of only one histidine which occupies the 95th position.     

ISOLATION AND AMINO ACID COMPOSITION OF SAUVAGINE

The skin of the South American frog Phyllomedusa sauvagei contains a new active polypeptide, sauvagine, which does not belong to any of the peptide families hitherto described in the amphibian skin. Sauvagine was isolated in a pure form starting from crude methanol extracts. The purification procedure involved several successive steps: dialysis, precipitation with ethanol and acetone, gel filtration and ion exchange chromatography. Two forms of sauvagine were separated. The major component (sauvagine I) was submitted to acid hydrolysis. The sauvagine molecule appeared to possess clear hydrophobic characteristics and to consist of a straight chain of 40 amino acid residues, among which the glutamyl-, aspartyl- leucyl- and isoleucyl-residues were particularly well represented. The elucidation of the amino acid sequence in the sauvagine molecule is in progress.     

ISOLATION AND CHARACTERIZATION OF THE CYANOGEN BROMIDE FRAGMENTS OF LOBSTER ARGININE KINASE (HOMARUS VULGARIS)

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10%) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to different gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position; therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

DETERMINATION OF THE COMPLETE AMINO ACID SEQUENCE OF BOVINE NEUROPHYSIN II

Bovine neurophysin II (BNP-II), a major neurohypophyseal hormone-binding protein in the cow, was isolated from acetone-dried posterior pituitary powder by gel filtration, ion exchange chromatography and preparative disc electrophoresis. The single-chain protein is composed of 97 amino acids, possesses a molecular weight of 10,029, and contains seven disulfide bonds. The fully S-alkylated protein, S-carboxamido-[C]- methylcysteine BNP-II ([C]BNP-II), was subjected to 80 automated cycles of the Edman degradation with positive identification of every amino acid residue through 65 cycles and many identifications through 80 residues. [C]BNP-II was digested with chymotrypsin and the peptides were isolated by gel filtration and purified by ion exchange chromatography. A 55-amino acid C-terminal chymotryptic peptide was sequenced through 46 residues by automated Edman degradations producing a 38-amino acid overlap with the sequence of [C]BNP-II. This established the sequences of the first 88 residues of the protein. A chymotryptic nonapeptide was sequenced from NH₂- to COOH-terminus by manual Edman degradations which established the sequence through residue 94. COOH-terminal analysis of aminoethylated BNP-II elucidated the tetrapeptide sequence and produced an overlap with the sequenced chymotryptic nonapeptide, completing the proposed amino acid sequence, which is supported by amino acid compositions of three other chymotryptic and two tryptic peptides.     

PRIMARY STRUCTURE OF EQUINE GROWTH HORMONE

The study of the structure of the peptides arising from native, oxidized, reduced and alkylated or citraconylated equine growth hormone on incubation with trypsin and chymotrypsin is reported. The data obtained, combined with results already published, support the assembly of a unique sequence of amino acids for the polypeptide chain of the protein.     

Bony fish neurophysins Identification of MSEL- and VLDV-neurophysins of the pollack (Pollachius virens)

The two types of neurophysins known in vertebrate species, namely MSEL-neurophysin (vasopressin-like hormone-associated neurophysin) and VLDV-neurophysin (oxytocin-like hormone-associated neurophysin) have been purified from the pollack (Pollachius virens) pituitary through a combination of molecular sieving and high-pressure liquid chromatography (HPLC). Homogeneity has been checked by gel electrophoresis and rerun in HPLC. The apparent molecular masses measured by SDS-electrophoresis are near 12 kDa, significantly higher than those found for their mammalian homologues (10 kDa). The two types of neurophysins have been recognized through their N-terminal amino acid sequences. The primary structure of MSEL-neurophysin has been partially determined using automated Edman degradation applied on native and reduced-alkylated protein, as well as peptides derived by trypsin or staphylococcal proteinase hydrolyses. Comparison of pollack MSEL-neurophysin with ox, goose and frog counterparts reveals that particular positions in the polypeptide chain are subjected to substitutions and that the numbers of substitutions do not seem closely related to the paleontological times of divergence between the different vertebrate classes.     

重组L-天门冬酰胺酶II的液相色谱/电喷雾离子化质谱法分析

目的　对基因重组L-天门冬酰胺酶II产品进行一级结构分析。方法　采用流动注射方式,电喷雾离子化质谱法测定分子量;三氯乙酸变性,胰蛋白酶水解后,HPLC测定肽图谱;结合质谱源内碰撞诱导解离技术,解析酶解肽段的氨基酸顺序。结果　分子量测定值与理论值不符,质谱解析表明样品中存在3个氨基酸变异现象,由此计算的分子量与测定值吻合。结论　液相色谱/电喷雾质谱法为基因重组蛋白质药物的一级结构分析和质量控制提供了新途径。     

REMOVAL OF PROTECTED PEPTIDES FROM THE MERRIFIELD RESIN BY POTASSIUM CYANIDE CATALYZED TRANSESTERIFICATION

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.     

ISOLATION AND CHARACTERIZATION OF CYANOGEN BROMIDE FRAGMENTS OF THE A AND B CHAINS OF THE ANTITUMOR TOXIN RICIN D

Both the A and B chains of ricin D consist of four CNBr fragments. These fragments have been isolated from the separated chains of S-carboxymethylated ricin D obtained from the small bean variety of Ricinus communis. The peptides have been characterized by molecular weight, amino acid composition and amino terminal sequence. A unique order of the peptides is evident for each chain. These sequence data are compared with those obtained for ricin D isolated from the large bean variety (Funatsu et al, 1979; Yoshitake et al., 1978).     

Partial amino acid sequence of porcine elastase II

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (M_r = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser¹⁹⁵. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.