Enzymatically catalyzed synthesis of oxytocin fragments 1-6 and 7-9

1985 ◽  
Vol 50 (12) ◽  
pp. 2775-2782 ◽  
Author(s):  
Václav Čeřovský ◽  
Karel Jošt
Keyword(s):  
Ethyl Ester ◽  
Sulfhydryl Group ◽  
Amino Groups ◽  
Equimolar Ratio ◽  
Peptide Bonds ◽  
The Given

Papain, α-chymotrypsin, thermolysin and elastase were utilized in the synthesis of peptide bonds of the protected oxytocin nonapeptide, except the S-benzylcysteine-proline bond. Amino groups were protected with benzyloxycarbonyl or tert-butyloxycarbonyl groups, carboxy groups as ethyl ester, phenylhydrazides or amides. The cysteine sulfhydryl group was blocked with the benzyl group whereas the tyrosine hydroxyl was unprotected. Most of the fragments were synthesized in satisfactory yields using an equimolar ratio of both reaction components and minimal (experimentally determined) amount of the given enzyme.

Synthesis of pressinoic acid by enzymatically catalyzed formation of peptide bonds

1986 ◽  
Vol 51 (6) ◽  
pp. 1352-1360 ◽  
Author(s):  
Václav Čeřovský
Keyword(s):  
Methyl Esters ◽  
Sulfhydryl Group ◽  
Carboxyl Groups ◽  
Amino Groups ◽  
Peptide Bonds ◽  
Fragment Condensation

Three fully enzymatic syntheses of the 1-6 vasopressin hexapeptide were investigated using papain, α-chymotrypsin and thermolysin. Best results were obtained with thermolysin in the 2 + 4 fragment condensation. The α-chymotrypsin-catalyzed 3 + 3 condensation is less advantageous and the 4 + 2 condensation with papain gave only low yield. Using the mentioned enzymes, further fragments of vasopressin molecule were prepared. Amino groups were protected with benzoylcarbonyl or tert-butyloxycarbonyl groups, carboxyl groups as phenylhydrazides or methyl esters, and the cysteine sulfhydryl group as the benzyl derivate. The tyrosine hydroxyl was not protected.

Weitere Untersuchungen zur Aminosäuresequenz des Melittins, II.

1969 ◽  
Vol 24 (4) ◽  
pp. 415-418 ◽  
Author(s):  
Joachim Jentsch
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Specificity ◽  
Bee Venom ◽  
Side Chains ◽  
Amino Groups ◽  
Crotalus Atrox ◽  
Peptide Bonds ◽  
Rattlesnake Venom

Degradation of melittin with α-Protease from Crotalus atrox venom (rattlesnake) confirmed the amino acid sequence of the toxic main peptide from bee venom. Hydrolysis occured mainly at the peptide bonds whose amino groups were provided by hydrophobic side chains such as valine, leucine and isoleucine bonds. However, on the contrary one glutamine bond was cleaved. Neverthelness, regarding the relatively high specificity, rattlesnake venom protease may be a valuable reagent for further sequence studies.

Evidence for Nonproductive Binding Subsites within the Active Site of Papain

1974 ◽  
Vol 52 (10) ◽  
pp. 877-883 ◽  
Author(s):  
Bernard R. Glick ◽  
Lewis J. Brubacher
Keyword(s):  
Ethyl Ester ◽  
Rate Constant ◽  
Active Site ◽  
Alkylating Agents ◽  
Sulfhydryl Group ◽  
Competitive Inhibitor ◽  
Alkylating Reagents ◽  
Nonproductive Binding ◽  
Binding Subsites

The reactions of several alkylating reagents with the sulfhydryl group in papain have been studied in the presence of varying concentrations of the competitive inhibitor α-N-benzoyl-D-arginine ethyl ester. The ratio of the alkylation rate constant of the papain – α-N-benzoyl-D-arginine ethyl ester complex to the rate constant with free papain is 4.3, 1.2, and 0.0 for the alkylating agents 1-chloro-3-tosylamido-4-phenyl-2-butanone, N-ethylmaleimide, and 1-chloro-3-tosylamido-7-amino-2-heptanone, respectively. These results are rationalized, along with data for the effect of α-N-benzoyl-L-arginine ethyl ester, in terms of nonproductive binding.

scholarly journals Oxidative modification of ovalbumin.

1996 ◽  
Vol 43 (4) ◽  
pp. 661-672 ◽  
Author(s):  
S Olszowski ◽  
E Olszowska ◽  
T Stelmaszyńska ◽  
A Krawczyk ◽  
J Marcinkiewicz ◽  
...  
Keyword(s):  
Active Oxygen Species ◽  
Sulfhydryl Group ◽  
Oxidative Modification ◽  
Oxidation Products ◽  
Amino Groups ◽  
Covalent Bonds ◽  
Mixed Oxidation ◽  
Sds Page ◽  
Hplc Profiles ◽  
Model Protein

Stimulated neutrophils (PMNL) are a source of the active oxygen species: O2, H2O2 and HOCl/OCl- which in turn can act on proteins yielding a variety of mixed oxidation products. A system is proposed in which a model protein-ovalbumin (OVA) first undergoes chlorination by HOCl/OCl- and next is oxidised by H2O2. The modification of functional groups (-NH2, -SH, -S-S-, > C = O, Tyr and Trp) in OVA was monitored as well as their accessibility to promote aggregation. Chlorination resulted in additional inter- or intra -S-S- bond formation followed by a decrease in the total sulfhydryl group content. Amino groups were oxidised to carbonyl moieties with a concomitant acidic shift of pI. Formation of chlorotyrosine at the chlorination step was confirmed and its further H2O2-mediated transformation to bityrosine was demonstrated. It has also been confirmed that tryptophan, and not tyrosine, is the first target for chlorination. SDS/PAGE and HPLC profiles revealed that HOCl/OCl- chlorination promotes formation of aggregates stabilised by non covalent bonds. In conclusion, we suggest that a dramatic change in the OVA molecule structure begins when the molar excess of HOCl/OCl- is about 2 per one reactive group in OVA.

THE SELECTIVE DEGRADATION OF WHEAT GLUTEN

1955 ◽  
Vol 33 (8) ◽  
pp. 1295-1303 ◽  
Author(s):  
L. Wiseblatt ◽  
L. Wilson ◽  
W. B. McConnell
Keyword(s):  
Strong Association ◽  
Average Molecular Weight ◽  
Wheat Gluten ◽  
Strong Acid ◽  
Terminal Group ◽  
Pressure Measurements ◽  
Amino Groups ◽  
Peptide Bonds ◽  
Selective Degradation ◽  
Chemical Cross Linking

A method believed to hydrolyze peptide bonds of proteins selectively at the amino groups of serine was used to obtain polypeptides from wheat gluten. The procedure involved the use of strong acid and introduced appreciable amounts of sulphur into the products possibly as sulphonic acid groups. Most of the serine appeared at the amino termini of the peptides. The peptides displayed a striking electrophoretic homogeneity which may at least in part be accounted for by the acquired acid groups. Osmotic pressure measurements indicated an average molecular weight near 20,000 and terminal group estimates indicate that each molecule contained several N-terminal serine residues. There appeared to be strong association or chemical cross linking between peptide chains of the degraded gluten.

ANTIGENICITY OF 5-HYDROXYINDOLE-3-ACETIC ACID, A DERIVATIVE OF SEROTONIN: I. PREPARATION OF PROTEIN CONJUGATES OF 5-HYDROXYINDOLE-3-ACETIC ACID

1967 ◽  
Vol 45 (11) ◽  
pp. 1681-1688 ◽  
Author(s):  
N. S. Ranadive ◽  
A. H. Sehon
Keyword(s):  
Acetic Acid ◽  
Aminocaproic Acid ◽  
Acid Anhydride ◽  
Rabbit Serum ◽  
Amino Groups ◽  
Serum Albumins ◽  
Peptide Bonds ◽  
Protein Conjugates ◽  
Γ Globulins

5-Hydroxyindole-3-acetic acid (5HIAA) was coupled to human, bovine, and rabbit serum albumins, to bovine γ-globulins, and to ε-aminocaproic acid (ACA) through the formation of peptide bonds. For this purpose N,N′-dicyclohexylcarbodiimide was used to prepare the intermediate acid anhydride of 5HIAA, which was subsequently reacted with the amino groups of proteins or ACA. Evidence is presented for the coupling of (i) about 18 residues of 5HIAA per molecule of albumin, (ii) about 30 residues per globulin molecule, and (iii) 1 residue per molecule of ACA.

scholarly journals THE ACTION OF ENZYMES ON RHODOPSIN

1958 ◽  
Vol 42 (2) ◽  
pp. 371-383 ◽  
Author(s):  
Charles M. Radding ◽  
George Wald
Keyword(s):  
Pancreatic Lipase ◽  
Cage Effect ◽  
Enzyme Molecule ◽  
Amino Groups ◽  
Peptide Bonds ◽  
Rhodopsin Molecule ◽  
Solvent Molecules ◽  
Rapid Hydrolysis ◽  
Two Stages ◽  
Hydrolysis Of

The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties.

THE ACTION OF SULPHURIC ACID ON GLIADIN: WITH SPECIAL REFERENCE TO THE N-PEPTIDYL→O-PEPTIDYL BOND REARRANGEMENT

1955 ◽  
Vol 33 (11) ◽  
pp. 1638-1648 ◽  
Author(s):  
L. K. Ramachandran ◽  
W. B. McConnell
Keyword(s):  
Sulphuric Acid ◽  
Direct Evidence ◽  
Acyl Migration ◽  
Hydroxyl Group ◽  
Amino Groups ◽  
Peptide Bonds ◽  
Alternative Scheme ◽  
Apparent Homogeneity ◽  
Secondary Reactions ◽  
Bond Rearrangement

Treatment of gliadin with sulphuric acid transposes peptide bonds of serine from the amino to the hydroxyl group. Maximum transposition, 60–70% of the theoretical, occurs when the protein is treated with anhydrous sulphuric acid at 0°C. for 35 hr. No rearrangement was detected at threonine residues. Examination of the peptide material, obtained from the rearranged protein by Elliott's degradation method, indicates apparent "homogeneity". In an alternative scheme for the degradation, nitrous acid deamination of free amino groups was used. The resulting loss in serine content of the protein is direct evidence for the acyl migration of peptide bonds. Incorporation of sulphur and partial disappearance of several amino acids accompany the sulphuric acid treatment. The occurrence of these secondary reactions imposes limitations on the use of sulphuric acid as a reagent for the specific fission of peptide bonds.

Coupling of Amino Acids and Amino Sugars with Cyanuric Chloride (2,4,6-Trichloro-s-triazine)

1972 ◽  
Vol 50 (13) ◽  
pp. 1987-1991 ◽  
Author(s):  
A. S. Chaudhari ◽  
C. T. Bishop
Keyword(s):  
Amino Acids ◽  
Ethyl Ester ◽  
Cyanuric Chloride ◽  
High Yield ◽  
Amino Sugars ◽  
Amino Groups ◽  
Mild Conditions ◽  
Glycine Ethyl Ester

2,4,6-Trichloro-s-triazine (cyanuric chloride) has been used to couple glycine ethyl ester with seven carbohydrate derivatives that contain amino groups. The reactions proceed in high yield under mild conditions. The compounds serve as models for the coupling of carbohydrates to proteins by well-defined linkages through amino groups.

Aldehyde gold clusters for molecular labeling

1995 ◽  
Vol 53 ◽  
pp. 858-859
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya
Keyword(s):  
Covalent Bond ◽  
Aldehyde Group ◽  
Gold Clusters ◽  
Side Reactions ◽  
Amino Groups ◽  
Sodium Cyanoborohydride ◽  
Schiff’S Base ◽  
Protein Molecules ◽  
Hydroxysuccinimide Esters ◽  
Primary Amino

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

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