Complete amino acid sequences of two cardiotoxin-like analogues from Bungarus fasciatus (banded krait) snake venom

Three cardiotoxin-like proteins have been isolated from Bungarus fasciatus venom and the amino acid sequence of the major toxin (toxin VI) have been determined. The amino acid sequences of two other analogues (toxins V-2 and V-3) were investigated. The reduced and S-carboxymethylated toxins were digested with trypsin-TPCK and the resulting tryptic peptides were isolated by fingerprinting technique on paper. The amino acid compositions, N-terminal residues and partial amino acid sequences of some of the tryptic peptides were determined. Thirteen tryptic peptides were aligned by following the order of corresponding fragments of toxin VI. Toxins V-2 and V-3 contained 118 and 117 amino acid residues, respectively, in a single polypeptide chain cross-linked with six pairs of intramolecular disulphide bonds. There are only four (for toxin V-2) and five (for toxin V-3) places of differences in their primary structures when compared with that of the major toxin (toxin VI) of Bungarus fasciatus venom.     

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 sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis australis)

Two basic phospholipases A2 (Pa-11 and Pa-13) have been isolated from the venom of an Australian elapid snake, Pseudechis australis (king brown snake). The reduced and S-carboxymethylated phospholipases A2 were digested with trypsin and the resulting peptides were purified by a combination of chromatography on a DEAE-cellulose DE-52 column and gel filtration procedures. Eleven main peptides from Pa-11 and 9 peptides from Pa-13 could account for the amino acid compositions of the respective enzyme molecules. The alignment of the tryptic peptides and unelucidated regions of the amino acid sequences of tryptic peptides were established by the analysis of the peptides obtained by chymotryptic and/or Staphylococcal protease digestions. Each phospholipase A2 consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. Although Pa-11 is enzymatically 30-times as active as Pa-13 and highly toxic as compared to Pa-13, they are highly homologous in their amino acid sequences. They are also homologous to the enzymes from mammalian pancreas and the other snake venom phospholipases A2, especially to those from snakes belonging to the subfamilies Acanthophiinae and Laticaudinae.     

Detection of desamido forms of purified bovine growth hormone

Tryptic peptide mapping and amino acid sequencing were used for the identification of desamido forms of bovine growth hormone. The major desamido forms of bovine growth hormone resulted from deamidation of asparagine in position 13, 148 and glutamine 140. These forms did not seem to be produced by prolonged treatment of bovine growth hormone in tryptic hydrolysis.     

Primary structure of elephant pituitary prolactin

Tryptic digests of elephant pituitary prolactin (elePRL) were separated by reverse phase high performance liquid chromatography (HPLC) and paper electrophoresis. From the amino acid composition, the amino acid sequencing of selected peptides, and from their alignment with expected tryptic peptides from ovine prolactin (oPRL), the primary sequence of elePRL is proposed.     

Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery

There are many examples of biologically active food proteins, with physiological significance beyond the pure nutritional requirements that concern available nitrogen for normal growth and maintenance. Moreover, there are many physiologically active peptides, derived by protease activity from various food protein sources; however, relationships between structural properties and functional activities have not been completely elucidated. Many bioactive peptides have in common structural properties that include a relatively short peptide residue length (e.g. 2-9 amino acids), possessing hydrophobic amino acid residues in addition to proline, lysine or arginine groups. Bioactive peptides are also resistant to the action of digestion peptidases. Antihypertensive peptides, known as Angiotensin I converting enzyme (ACE) inhibitors have been derived from milk, corn and fish protein sources. Peptides with opioid activities are derived from wheat gluten or casein, following digestion with pepsin. Exorphins, or opioid peptides derived from food proteins such as wheat and milk (e.g. exogenous sources) have similar structure to endogenous opioid peptides, with a tyrosine residue located at the amino terminal or bioactive site. Immunomodulatory peptides derived from tryptic hydrolysates of rice and soybean proteins act to stimulate superoxide anions (reactive oxygen species-ROS), which triggers non-specific immune defense systems. Antioxidant properties that prevent peroxidation of essential fatty acids have also been shown for peptides derived from milk proteins. The addition of a Leu or Pro residue to the N-terminus of a His-His, dipeptide will enhance antioxidant activity and facilitate further synergy with non-peptide antioxidants (e.g. BHT). We also show herein, that the tryptic digests of casein yielding caseinophosphopeptides exhibits both hydrophilic and lipophilic antioxidant activity due to both metal ion sequestering and quenching of ROS. The separation and purification of bioactive peptides which will involve development of automated and continuous systems is an important field for Food chemists. Much effort has been given to develop selective column chromatography methods that can replace batch methods of salting out, or using solvent extraction to isolate and purify bioactive peptides. Advances here will enable recovery of bioactive peptides with minimal destruction thus enabling utilization by returning these active peptides to functional food or specific nutraceutical applications.     

Primary structure of alpaca growth hormone

Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptides in the mixture were purified by reverse phase HPLC and N-terminal determination and an amino acid analysis of each was performed. Data obtained and the already known primary structure of the equine growth hormone allowed the assembly-by homology-of a definite sequence of amino acids for the polypeptide chain of the protein. Present data provide further information about the relationship between growth factors.     

Complete amino acid sequence of a phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The acidic phospholipase A₂ has been isolated from Naja naja atra (Taiwan cobra) venom. The reduced and S-carboxymethylated enzyme was digested with trypsin, and eleven tryptic peptides which accounted for the whole molecule were isolated by a combination of chromatographic and gel filtration procedures, and their amino acid sequences were determined. The alignment of those eleven tryptic peptides was established by the analysis of nine major peptides obtained by Staphylococcus aureus protease digestion of the reduced and S-carboxymethylated enzyme. The phospholipase A₂ of Naja naja atra is a single polypeptide chain consisting of 120 amino acid residues including 14 intramolecularly linked half-cystines. It shows about 86% (103 out of 120) homology when compared with the Naja mossambica mossambica CM-II enzyme.     

FACILE ASSIGNMENT OF DISULFIDE BONDS IN OVINE LACTOGENIC HORMONE AND HUMAN GROWTH HORMONE

A procedure, recently developed in this laboratory, for the rapid determination of the positions of disulfide bonds in proteins of known amino acid sequence was applied to the assignment of the disulfide bonds of ovine lactogenic hormone and human growth hormone. Each protein was hydrolysed by pepsin under conditions where disulfide interchange does not occur. Peptide maps of the resulting hydrolysates were prepared. Sodium borohydride reduction and subsequent treatment with S,S'-dithiobis (2-nitrobenzoic acid) (DNTB) revealed the locations of cystine-containing peptides: four for ovine lactogenic hormone and three for human growth hormone. These cystine-containing peptides were eluted, hydrolysed, and their amino acid compositions determined. By comparison of the analytical data with the known amino acid sequences around each half-cystine residue and the known substrate specificity of pepsin, the positions of the disulfide bridges were unequivocally determined. The results confirm the disulfide linkages previously established by other methods. The scope and limitations of this procedure are discussed.     

Amino acid sequence of pelamitoxin a, the main neurotoxin of the sea snake, Pelamis platurus

The amino acid sequence of pelamitoxin a, the main neurotoxin of the sea snake, Pelamis platurus, has been completed. Analysis was done principally by means of Edman degradation of constituent tryptic peptides; chymotryptic and thermolytic peptides were also studied to assist in the sequence alignment. The results indicate that pelamitoxin a is identical to schistosa toxin 5 in amino acid sequence. These results indicate the feasibility of producing a common antivenin against toxins from some of the most common sea snakes.     

Use of proline-specific endopeptidase in the isolation of all four “native” disulfides of hen egg white lysozyme

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I–VIII, II–VII, and III–V + IV–VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE)(EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV–VI disulfide bond as well as a peptide with the III–V disulfide bond. The IV–VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV–VI and III–V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.     

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。     

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.     

STUDIES ON A PROTAMINE (GALLINE) FROM FOWL SPERM.

Of the eight fractions (G-I-G-VIII) obtained from galline, a protamine from rooster sperm nuclei, the complete amino acid sequences of two fractions G-I and G-V have already been reported. Other fractions of galline purified repeatedly on the column of Bio-Gel CM-30 were submitted to structural analyses. The complete amino acid sequences of three (G-II, G-III and G-VII) of the fractions and the partial chemical structures of two (G-VI and G-VIII) of the fractions were established from the results of analyses of the tryptic and chymotryptic peptides together with those of end groups and terminal sequences. Remarkable similarities have so far been found in the sequences of these fractions. Combining these results with those complementarily explained in the following paper, it was finally confirmed that the intact galline molecule is composed of only one molecular species (G-VIII) and that fractions (G-I-G-VII) were derived from G-VIII by the action of an inherent protease in the preparation process. Consequently, the total amino acid sequence of the intact galline is concluded to be as follows: H-Ala-Arg-Tyr-Arg-Ser-Arg-Gly-Arg-Ser-Arg-Ser-Arg-Arg-Thr-Arg-Arg-Arg-Arg-Ser-Pro-Arg-Ser-Gly-Arg-Arg-Arg-Ser-Pro-Arg-Arg-Arg-Arg-Ser-Arg-Arg-Arg-Arg-Arg-Tyr-Gly-Ser-Ala-Arg-Arg-Ser-Arg-Arg-Ser-Gly-Gly-Val-Arg-Arg-Arg-Arg-Tyr-Gly-Ser-Arg-Arg-Arg-Arg-Arg-Arg-65Tyr-OH.     

The parathyroid hormone, its fragments and analogues--potent bone-builders for treating osteoporosis

As populations age a rising number of men and women, but especially women during the first decade after menopause, become victims of a severe, accelerated loss of bone with crippling fractures known as osteoporosis. This often results in costly, prolonged hospitalisation and perhaps indirectly, death. Osteoporosis in women is caused by the menopausal oestrogen decline, which removes several key restraints on the generation, longevity and activity of bone-resorbing osteoclasts. Although there are many antiresorptive drugs on or coming onto the market (calcitonin, bisphosphonates, oestrogen and SERMS) that can slow or stop further bone loss, there are none that can restore lost bone mechanical strength by directly stimulating osteoblast activity and bone growth. However, there is a family of potent bone-building peptides, namely the 84 amino acid parathyroid hormone (PTH). Its 31 to 38 amino acid N-terminal fragments are currently in or about to enter clinical trials. We can predict that these peptides will be effective therapeutics for osteoporosis especially when supplemented with bisphosphonates or SERMs to protect the new bone from osteoclasts. These peptides should also accelerate the healing of fractures in persons of all ages and restore lost bone mass and mechanical strength to astronauts following their return to earth after long voyages in space.     

The parathyroid hormone,its fragments and analogues - potent bone-builders for treating osteoporosis

As populations age a rising number of men and women, but especially women during the first decade after menopause, become victims of a severe, accelerated loss of bone with crippling fractures known as osteoporosis. This often results in costly, prolonged hospitalisation and perhaps indirectly, death. Osteoporosis in women is caused by the menopausal oestrogen decline, which removes several key restraints on the generation, longevity and activity of bone-resorbing osteoclasts. Although there are many antiresorptive drugs on or coming onto the market (calcitonin, bisphosphonates, oestrogen and SERMS) that can slow or stop further bone loss, there are none that can restore lost bone mechanical strength by directly stimulating osteoblast activity and bone growth. However, there is a family of potent bone-building peptides, namely the 84 amino acid parathyroid hormone (PTH). Its 31 to 38 amino acid N-terminal fragments are currently in or about to enter clinical trials. We can predict that these peptides will be effective therapeutics for osteoporosis especially when supplemented with bisphosphonates or SERMs to protect the new bone from osteoclasts. These peptides should also accelerate the healing of fractures in persons of all ages and restore lost bone mass and mechanical strength to astronauts following their return to earth after long voyages in space.     

Equine growth hormone Detection of immunoreactive sequences using poly- and monoclonal antibodies

The immunochemical behavior of several fragments of equine growth hormone (eGH) was examined using competitive binding assays with antibodies (Abs) to eGH obtained from different sources. Antigenicity was detected within the sequences 5–72 and 73–123 by rabbit Abs to eGH and by three mouse monoclonal antibodies (MAbs) produced by using bovine growth hormone as immunogen, but showing heteroclitic properties towards eGH. The polyclonal Abs to eGH also recognized as immunoreactive two smaller peptides corresponding to the amino acid residues 52–72 and 110–123. By contrast, the heteroclitic Abs to eGH developed by hypopituitary patients therapeutically injected with human growth hormone failed to react with any eGH-derived fragment. The rabbit polyclonal Abs and the mouse MAbs scarcely discriminated between native and S-carbamidomethylated eGH, while the heteroclitic human Abs detected a clear difference between the native and the modified hormone.     

Nerve growth factor from Vipera lebetina venom

Nerve growth factor was isolated from the Vipera lebetina venom by a four-step procedure including gel filtration, ion exchange, heparin and hydrophobic chromatography. The purified protein is a glycosylated non-covalently bound homodimer with monomeric molecular mass of 14,380 Da. The cDNA encoding NGF is cloned and sequenced. The amino acid sequence translated from the cDNA comprises 117 or 119 amino acids depending on the N-terminus (truncated or not). The recombinant NGF (expressed in Escherichia coli) was used to prepare the anti-NGF antiserum. The antiserum interacted with the wild-type NGF and enabled to localize NGF during the purification procedure in parallel with MALDI-TOF analysis of tryptic peptides. The isolated NGF caused neurite outgrowth from PC12 cells in concentrations beginning from 2.5 ng/ml.     

Complete amino acid sequences of two protease inhibitors in the venom of Bungarus fasciatus

Two analogous protease inhibitors, VIIIb and IX in the venom of Bungarus fasciatus were reduced and carboxymethylated. Tryptic peptides were separated by cellulose thin-layer peptide mapping technique, and amino acid sequences were analyzed by DABITC/PITC double coupling method. Alignment of all tryptic peptides was established by analyses of chymotryptic peptides and further confirmed by Staphylococcus aureus V8 protease digestion. IX consisted of 65 amino acid residues. VIIIb consisted of 62 residues, identical to the N-terminal 62-amino acid sequence of IX.     

COMPUTER PROGRAM FOR LOCALIZATION OF DISULFIDE BONDS IN PROTEINS

A Fortran computer program to aid in disulfide bond analysis in proteins has been developed. The program evaluates amino acid analysis data from isolated cystine-containing peptides by comparing it with known sequences around pairs of half-cystine residues in the primary structure of a protein and recording all matches. The method involves presetting upper and lower bounds for each amino acid present in a cystine-containing peptide by considering both partial degradation during acid hydrolysis and contamination sources for certain amino acids. Initially, limiting path terminals for each half-cystine residue in the primary structure are determined by examining the surrounding sequences in both directions for “alien” amino acid residues, not present in the composition of a given cystine-containing peptide. Subsequently, pairwise examination proceeds through several cycles incorporating minimal path and upper bound routines with intermittent revision of path ends. Finally, all pairs of half-cystines whose surrounding sequences fall within the preset bounds for this peptide are listed. If too many possible pairs or none are found, the bounds are revised. The lists for all available cystine-containing peptides are graphed to facilitate final determination of disulfide bond positions in the native protein. The computer program provided finite disulfide bond assignments for ovine lactogenic hormone and human growth hormone. In the case of hen egg-white lysozyme, a considerable reduction was obtained from the large number of theoretically possible disulfide bond combinations to a few alternatives, and one of the four disulfide bonds was unequivocally determined. To assign the positions of the other three disulfide bridges, additional considerations, not included in the program, were applied, such as specificity of the enzymes used in digesting the protein and electrophoretic mobility of the ensuing cystine-containing peptides