Scandium(iii) triflate-promoted serine/threonine-selective peptide bond cleavage

Abstract The conditions used to hydrolyze proteins are vital in determining amino acid compositions because they necessarily represent a compromise aimed at yielding the best estimate of amino acid composition. Variations in ease of peptide bond cleavage, differences in amino acid stabilities, and matrix effects from nonproteinaceous components all militate against a single set of hydrolysis conditions that quantitatively hydrolyze every peptide bond and concurrently cause no destruction of any amino acid. This presentation summarizes and reviews an extensive study which evaluated a number of variations in the techniques and procedures of the classical 6N HC1, 110°C, 24 h hydrolysis of protein. The objectives of the recent investigation were: (/) to compare hydrolysis at 145°C, 4 h with 110°C, 24 h for proteins in a wide range of different sample matrixes; (2) to compare protein hydrolysis at 110°C, 24 h conducted in sealed glass ampoules after vacuum removal of air with hydrolysis in glass tubes with Teflon-lined screw caps after removal of air by vacuum, nitrogen purge, and sonication; (3) to evaluate a performic acid oxidation procedure before hydrolysis for the analysis of cystine and methionine in the different sample matrixes; (4) to evaluate multiple hydrolysis times at 145°C; (5) to evaluate the variation of interlaboratory hydrolysates prepared at 145°C, 4 h in 2 different laboratories on the amino acid analysis of an array of protein-containing matrixes. The major sources of inaccuracy and lack of precision arising from the application of ion-exchange or gas chromatography, both of which provide excellent accuracy and precision, are prechromatographic sample handling and the method used for hydrolysis of the protein sample itself. Hydrolysate preparation is the area that requires the most attention to solve problems of variability of amino acid analyses.

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Chemical modification of peptides by hydrazine

Biochemical Journal ◽

10.1042/bj1990053 ◽

1981 ◽

Vol 199 (1) ◽

pp. 53-59 ◽

Cited By ~ 18

Author(s):

A Honegger ◽

G J Hughes ◽

K J Wilson

Keyword(s):

High Pressure ◽

Solid Phase ◽

Bond Cleavage ◽

Low Cost ◽

Peptide Bond ◽

Reaction Products ◽

Reverse Phase ◽

Peptide Bonds ◽

Peptide Bond Cleavage ◽

Arginine Residues

High pressure (‘performance’) liquid chromatography on reverse-phase supports has been used to characterize the products arising from the hydrazine treatment of peptides. In addition to converting arginine residues into ornithine, the reaction was found to cleave predominately Gly-Xaa, Xaa-Gly, Asn-Xaa and Xaa-Ser peptide bonds. Peptide-bond cleavage and deguanidation was studied as a function of time of exposure to hydrazine, hydrazine concentration and temperature. The convenience of this method of chromatography for the rapid low-cost separation and isolation of peptides, as well as their reaction products, is illustrated at the level of material required for solid-phase microsequencing.

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Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

Scientific Reports ◽

10.1038/s41598-021-88432-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Riley B. Peacock ◽

Taylor McGrann ◽

Marco Tonelli ◽

Elizabeth A. Komives

Keyword(s):

Active Site ◽

Serine Proteases ◽

Protein C ◽

Catalytic Mechanism ◽

Bond Cleavage ◽

Peptide Bond ◽

Peptide Bond Cleavage ◽

Exchange Mass Spectrometry ◽

Catalytically Active ◽

Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

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Activation of Bovine Factor V by Thrombin and A Protease From Russell's Viper Venom (RVV)

10.1055/s-0039-1687642 ◽

1979 ◽

Author(s):

M.J. Lindhout ◽

C. M. Jackson

Keyword(s):

Bond Cleavage ◽

Peptide Bond ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Factor V ◽

Peptide Bond Cleavage ◽

Prothrombinase Complex ◽

Factor Va ◽

Activation Products ◽

Ca Oxalate

In order to understand the function of activated factor V in the prothrombinase complex, we isolated the activation products obtained by action of thrombin and RVV-V on factor V and studied their functional properties. Factor V isolated from plasma by means of ion-exchange chromatography, a Ca-oxalate adsorption step and gelfiltration was homogenous in SDS-gelelectrophoresis (apparent MW 360,000, with and without reduction). Increase in factor V activity upon action by RVV-V is correlated with a single peptide bond cleavage, resulting in a 270,000 dalton and a 80,000 dalton component. Additional proteolysis of factor Va(RVV/V)’ by thrombin results in a further cleavage of the high MW component into peptides with MW's of 72,000, 94,000 and about 150,000 without a furth~r increase in factor V activity. Whereas none of the isolated peptides reveal factor Va activity, activity would be generated by a recombination in the presence of Ca2+ of the 94,000 MW or 270,000 MW component with the 80,000 component. Action of thrombin alone on factor V results in peptides of MW 72,000, 80,000, 94,000 and a peptide very rich in carbohydrate with an apparent MW of 150,000.

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The Protein Core of the Largest Proteoglycan Monomer of Articular Cartilage. Gel Electrophoretic Pattern and Tryptophanyl Peptide Bond Cleavage

Connective Tissue Research ◽

10.3109/03008208709005622 ◽

1987 ◽

Vol 16 (4) ◽

pp. 377-384 ◽

Cited By ~ 1

Author(s):

Victor Stanescu ◽

Françoise Chaminade ◽

Marie-Paule Muriel

Keyword(s):

Articular Cartilage ◽

Bond Cleavage ◽

Electrophoretic Pattern ◽

Peptide Bond ◽

Protein Core ◽

Peptide Bond Cleavage

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Isolation, purification and structure of exochelin MS, the extracellular siderophore from Mycobacterium smegmatis

Biochemical Journal ◽

10.1042/bj3050187 ◽

1995 ◽

Vol 305 (1) ◽

pp. 187-196 ◽

Cited By ~ 62

Author(s):

G J Sharman ◽

D H Williams ◽

D F Ewing ◽

C Ratledge

Keyword(s):

Amino Acids ◽

Mycobacterium Smegmatis ◽

Methyl Esters ◽

Three Dimensional ◽

Iron Chelation ◽

Peptide Bond ◽

Exchange Chromatography ◽

Peptide Bonds ◽

Hydrolysis Of ◽

Gallium Complex

The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to > 98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9.5), has a lambda max at 420 nm and a probable Ks for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY and TOCSY) 1H n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl) -beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.

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