scholarly journals Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination

1981 ◽  
Vol 199 (2) ◽  
pp. 335-340 ◽  
Author(s):  
G C Dubois ◽  
E A Robinson ◽  
J K Inman ◽  
R N Perham ◽  
E Appella
Keyword(s):  
Amino Acid ◽  
Primary Structure ◽  
Half Life ◽  
Structural Studies ◽  
Amino Groups ◽  
Mouse Immunoglobulin ◽  
Lysine Residues ◽  
Rapid Removal ◽  
Proteins And Peptides

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

Determination of Factor Xa Activity by Means of Chromogenic Substrates Based on the Primary Structure of Prothrombin

1975 ◽  
Author(s):  
L. Aurell ◽  
A. Olausson ◽  
G. Claeson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Serine Proteases ◽  
Factor Xa ◽  
Amino Acid Sequences ◽  
Peptide Substrate ◽  
Chromogenic Substrates ◽  
Special Regard

Through the work of Magnusson and co-workers leading to the elucidation of the primary structure of prothrombin including the amino acid sequences around the two bonds split by factor Xa it has been possible to design a synthetic chromogenic peptide substrate. Bz-Ile-Glu-Gly-Arg-pNA, specifically intended for the determination of factor Xa. Furthermore, additional substrates have been synthezised with various alterations in the amino acid sequence. The activity of factor Xa and other serine proteases within the coagulation and fibrinolytic systems towards these substrates will be discussed with special regard to their possible use in coagulation studies.

scholarly journals Generation of Affinity Purified Antibody Reagents for Specific Determination of Efruxifermin in Biological Matrices

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A288-A288
Author(s):  
Adam S Kinne ◽  
Sanofar J Abdeen ◽  
Elijah S Parmer ◽  
Jennifer A Thystrup ◽  
Erik J Tillman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Half Life ◽  
Clinical Development ◽  
Amino Acid Sequences ◽  
Fibroblast Growth Factor 21 ◽  
Biological Matrices ◽  
Specific Determination ◽  
Human Igg1 ◽  
Human Fgf21

Abstract Efruxifermin (EFX) is a novel Fc-fusion analog of human fibroblast growth factor 21 (FGF21), currently in clinical development as a potential treatment for non-alcoholic steatohepatitis (NASH). Each molecule of EFX consists of two modified FGF21 molecules, each attached at their N-termini to a human IgG1 Fc domain by a short polyglycine-serine linker. The FGF21 moiety of EFX incorporates three amino acid substitutions (L98R, P171G, and A180E relative to native FGF21). Two of these are proximal to the C-terminus (P171G and A180E), and reduce cleavage and inactivation by an endogenous protease, fibroblast activation protein (FAP), thereby prolonging its half-life. Fusion to human IgG1 Fc domain further extends circulating half-life, enabling once-weekly subcutaneous dosing. Accordingly, to support on-going clinical development of EFX, a specific assay is needed to distinguish intact EFX from both endogenous FGF21 and any in vivo biotransformation products of EFX that display reduced pharmacology. To maximize the antigenicity of EFX, FGF21 amino acid sequences were compared across species. Based on this, an antibody generation campaign was initiated in both rabbits and chickens. Comparison of titer responses against EFX and human FGF21 suggested that antisera from chickens was superior to rabbit antisera. Following a scaled-up, 12-week antibody campaign, antisera were purified by a combination of batch and column chromatographic procedures. By exploiting differences in structure and amino acid sequence of EFX relative to human FGF21, a purification strategy was designed to isolate chicken antibodies with increased specificity for EFX unique sequences. This reagent is being used as a capture antibody in the development of a noncompetitive ECLIA employing chemiluminescence detection. Presently, a number of different antibodies are being evaluated for potential pairing with the specific capture. We conclude that application of affinity purified chicken anti-EFX IgY will enable sensitive and specific determination of EFX in biological matrices with decreased cross-reactivity from endogenous hFGF21 and EFX metabolites.

REACTION OF FORMALDEHYDE AND THIOFORMALDEHYDE WITH MERCURIPAPAIN

1965 ◽  
Vol 43 (7) ◽  
pp. 1171-1177 ◽  
Author(s):  
W. A. Darlington ◽  
L. Keay
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Functional Groups ◽  
Tertiary Structure ◽  
Colorimetric Assay ◽  
Amino Groups ◽  
Full Activity ◽  
Amino Acid Side Chains ◽  
Lysine Residues ◽  
Secondary And Tertiary Structure

In a colorimetric assay with benzoyl-DL-arginine p-nitroanilide, acetylated and benzoylated papains retain full activity. Thus the ε-amino groups of the lysine residues are not required for enzyme activity. Intramolecular crosslinking of an enzyme could in theory stabilize secondary and tertiary structure and oppose denaturation. Thioformaldehyde is much more reactive with mercuripapain than formaldehyde, incorporating much more readily into the enzyme at equivalent concentrations. Incorporation is extensive, however, on reactive functional groups on the amino acid side chains, since acetylation decreased the incorporation markedly. In no case was there evidence of heat stabilization.

scholarly journals The primary structure of histone H2A from the nematode Caenorhabditis elegans

1987 ◽  
Vol 243 (1) ◽  
pp. 297-300 ◽  
Author(s):  
J R Vanfleteren ◽  
S M Van Bun ◽  
J J Van Beeumen
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Primary Structure ◽  
Amino Acid Residues ◽  
Histone H2a ◽  
Nematode Caenorhabditis Elegans ◽  
Calf Thymus ◽  
N Terminus ◽  
Lysine Residues ◽  
Complete Primary Structure

The complete primary structure of histone H2A from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 126 amino acid residues and has a blocked N-terminus. By comparison with calf thymus histone H2A, the nematode protein shows five deletions, two insertions and 16 substitutions. Most of the changes occur in the N- and C-terminal regions of the molecule, whereas the central part covering the residues 21-120 is quite well conserved. The lysine residues 5, 8 and 10 were found to be partially acetylated.

scholarly journals Primary structure of hemoglobins from trout(Salmo irideus). Partial determination of amino acid sequence of Hb trout IV

FEBS Letters ◽  
1976 ◽  
Vol 64 (1) ◽  
pp. 76-80 ◽  
Author(s):  
F. Bossa ◽  
D. Barra ◽  
M. Coletta ◽  
F. Martini ◽  
A. Liverzani ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure
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