Staphylococcus aureus V8-protease catalyzed segment exchange reaction of alpha-chain of hemoglobin S: a semisynthetic approach for the preparation of variants of alpha-chain.

A. S. Acharya, Y. J. Cho, K. S. Iyer

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Abstract

The Glu(30)-Arg(31) peptide bond of alpha-chain of hemoglobin is readily and quantitatively hydrolyzed by Staphylococcus Aureus V8-protease at pH 4.0 and 37 degrees C. This region of the alpha-chain represents a 'permissible discontinuity region' of the chain within its tertiary interactions. Protease catalyzed reformation of peptide bonds in such permissible discontinuity regions of fragment complementing systems of proteins appears to be a useful procedure for the preparation of variants of the protein. Therefore, attempts have been now made to 'restitch' the segments alpha 1-30 and alpha 31-141 using V8-protease. The alpha-amino group of alpha 1-30 was selectively dihydroxypropylated (DHP) using [14C]-glyceraldehyde to follow the protease catalyzed reformation of globin. V8-protease catalyzed the condensation of N alpha-DHP-alpha 1-30 and alpha 31-141 to generate N alpha-DHP-alpha 1 141 in the presence of 30% propanol at pH 6.0 and 4 degrees C. The synthetic yield of N alpha-DHP-alpha 1-141 is about 55% in 48 hrs and remained nearly the same even after seven days. Under the same conditions alpha-globin was digested at the Glu(30)-Arg(31) peptide bond to nearly 40%. Thus, the amount of Glu(30)-Arg(31) peptide bond formed in 48 hrs appears to be the result of an equilibrium in the V8-protease catalyzed hydrolytic and synthetic reactions. The reformation of Glu(30)-Arg(31) bond appears to be very selective. V8-protease did not catalyze the peptide bond formation in an equimolar mixture of alpha 31-141 with either alpha 1-23, alpha 1-27, or alpha 28-30 all of which have glutamic acid as the carboxy terminal residue just as in alpha 1-30. The high selectivity in the protease catalyzed formation of Glu(30)-Arg(31) peptide bond suggests that the segment alpha 1-30 of the intact alpha-globin could be exchanged with synthetic analogs of alpha 1-30 using V8-protease without actually isolating the fragment alpha 31-141. Incubation of [14C]-N alpha-DHP-alpha 1-30 with alpha-globin at pH 6.0 and 4 degrees C along with V8-protease in 30% n-propanol indeed resulted in the exchange of the segment alpha 1-30 of alpha-globin with [14C]-N alpha-DHP-alpha 1-30. This V8-protease catalyzed 'segment exchange reaction' should facilitate the preparation of alpha-chains with double mutations from the large number of naturally occurring alpha-chains with single mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

Original languageEnglish (US)
Pages (from-to)3-19
Number of pages17
JournalProgress in Clinical and Biological Research
Volume240
StatePublished - 1987
Externally publishedYes

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Sickle Hemoglobin
Staphylococcus aureus
alpha-Globins
Peptides
1-Propanol
Peptide Hydrolases
Glyceraldehyde
Mutation
Globins
glutamyl endopeptidase
Glutamic Acid
Hemoglobins
Proteins

ASJC Scopus subject areas

  • Medicine(all)

Cite this

@article{02674d915a314983bd348a6117dc29d5,
title = "Staphylococcus aureus V8-protease catalyzed segment exchange reaction of alpha-chain of hemoglobin S: a semisynthetic approach for the preparation of variants of alpha-chain.",
abstract = "The Glu(30)-Arg(31) peptide bond of alpha-chain of hemoglobin is readily and quantitatively hydrolyzed by Staphylococcus Aureus V8-protease at pH 4.0 and 37 degrees C. This region of the alpha-chain represents a 'permissible discontinuity region' of the chain within its tertiary interactions. Protease catalyzed reformation of peptide bonds in such permissible discontinuity regions of fragment complementing systems of proteins appears to be a useful procedure for the preparation of variants of the protein. Therefore, attempts have been now made to 'restitch' the segments alpha 1-30 and alpha 31-141 using V8-protease. The alpha-amino group of alpha 1-30 was selectively dihydroxypropylated (DHP) using [14C]-glyceraldehyde to follow the protease catalyzed reformation of globin. V8-protease catalyzed the condensation of N alpha-DHP-alpha 1-30 and alpha 31-141 to generate N alpha-DHP-alpha 1 141 in the presence of 30{\%} propanol at pH 6.0 and 4 degrees C. The synthetic yield of N alpha-DHP-alpha 1-141 is about 55{\%} in 48 hrs and remained nearly the same even after seven days. Under the same conditions alpha-globin was digested at the Glu(30)-Arg(31) peptide bond to nearly 40{\%}. Thus, the amount of Glu(30)-Arg(31) peptide bond formed in 48 hrs appears to be the result of an equilibrium in the V8-protease catalyzed hydrolytic and synthetic reactions. The reformation of Glu(30)-Arg(31) bond appears to be very selective. V8-protease did not catalyze the peptide bond formation in an equimolar mixture of alpha 31-141 with either alpha 1-23, alpha 1-27, or alpha 28-30 all of which have glutamic acid as the carboxy terminal residue just as in alpha 1-30. The high selectivity in the protease catalyzed formation of Glu(30)-Arg(31) peptide bond suggests that the segment alpha 1-30 of the intact alpha-globin could be exchanged with synthetic analogs of alpha 1-30 using V8-protease without actually isolating the fragment alpha 31-141. Incubation of [14C]-N alpha-DHP-alpha 1-30 with alpha-globin at pH 6.0 and 4 degrees C along with V8-protease in 30{\%} n-propanol indeed resulted in the exchange of the segment alpha 1-30 of alpha-globin with [14C]-N alpha-DHP-alpha 1-30. This V8-protease catalyzed 'segment exchange reaction' should facilitate the preparation of alpha-chains with double mutations from the large number of naturally occurring alpha-chains with single mutation.(ABSTRACT TRUNCATED AT 400 WORDS)",
author = "Acharya, {A. S.} and Cho, {Y. J.} and Iyer, {K. S.}",
year = "1987",
language = "English (US)",
volume = "240",
pages = "3--19",
journal = "Progress in Clinical and Biological Research",
issn = "0361-7742",
publisher = "John Wiley and Sons Inc.",

}

TY - JOUR

T1 - Staphylococcus aureus V8-protease catalyzed segment exchange reaction of alpha-chain of hemoglobin S

T2 - a semisynthetic approach for the preparation of variants of alpha-chain.

AU - Acharya, A. S.

AU - Cho, Y. J.

AU - Iyer, K. S.

PY - 1987

Y1 - 1987

N2 - The Glu(30)-Arg(31) peptide bond of alpha-chain of hemoglobin is readily and quantitatively hydrolyzed by Staphylococcus Aureus V8-protease at pH 4.0 and 37 degrees C. This region of the alpha-chain represents a 'permissible discontinuity region' of the chain within its tertiary interactions. Protease catalyzed reformation of peptide bonds in such permissible discontinuity regions of fragment complementing systems of proteins appears to be a useful procedure for the preparation of variants of the protein. Therefore, attempts have been now made to 'restitch' the segments alpha 1-30 and alpha 31-141 using V8-protease. The alpha-amino group of alpha 1-30 was selectively dihydroxypropylated (DHP) using [14C]-glyceraldehyde to follow the protease catalyzed reformation of globin. V8-protease catalyzed the condensation of N alpha-DHP-alpha 1-30 and alpha 31-141 to generate N alpha-DHP-alpha 1 141 in the presence of 30% propanol at pH 6.0 and 4 degrees C. The synthetic yield of N alpha-DHP-alpha 1-141 is about 55% in 48 hrs and remained nearly the same even after seven days. Under the same conditions alpha-globin was digested at the Glu(30)-Arg(31) peptide bond to nearly 40%. Thus, the amount of Glu(30)-Arg(31) peptide bond formed in 48 hrs appears to be the result of an equilibrium in the V8-protease catalyzed hydrolytic and synthetic reactions. The reformation of Glu(30)-Arg(31) bond appears to be very selective. V8-protease did not catalyze the peptide bond formation in an equimolar mixture of alpha 31-141 with either alpha 1-23, alpha 1-27, or alpha 28-30 all of which have glutamic acid as the carboxy terminal residue just as in alpha 1-30. The high selectivity in the protease catalyzed formation of Glu(30)-Arg(31) peptide bond suggests that the segment alpha 1-30 of the intact alpha-globin could be exchanged with synthetic analogs of alpha 1-30 using V8-protease without actually isolating the fragment alpha 31-141. Incubation of [14C]-N alpha-DHP-alpha 1-30 with alpha-globin at pH 6.0 and 4 degrees C along with V8-protease in 30% n-propanol indeed resulted in the exchange of the segment alpha 1-30 of alpha-globin with [14C]-N alpha-DHP-alpha 1-30. This V8-protease catalyzed 'segment exchange reaction' should facilitate the preparation of alpha-chains with double mutations from the large number of naturally occurring alpha-chains with single mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

AB - The Glu(30)-Arg(31) peptide bond of alpha-chain of hemoglobin is readily and quantitatively hydrolyzed by Staphylococcus Aureus V8-protease at pH 4.0 and 37 degrees C. This region of the alpha-chain represents a 'permissible discontinuity region' of the chain within its tertiary interactions. Protease catalyzed reformation of peptide bonds in such permissible discontinuity regions of fragment complementing systems of proteins appears to be a useful procedure for the preparation of variants of the protein. Therefore, attempts have been now made to 'restitch' the segments alpha 1-30 and alpha 31-141 using V8-protease. The alpha-amino group of alpha 1-30 was selectively dihydroxypropylated (DHP) using [14C]-glyceraldehyde to follow the protease catalyzed reformation of globin. V8-protease catalyzed the condensation of N alpha-DHP-alpha 1-30 and alpha 31-141 to generate N alpha-DHP-alpha 1 141 in the presence of 30% propanol at pH 6.0 and 4 degrees C. The synthetic yield of N alpha-DHP-alpha 1-141 is about 55% in 48 hrs and remained nearly the same even after seven days. Under the same conditions alpha-globin was digested at the Glu(30)-Arg(31) peptide bond to nearly 40%. Thus, the amount of Glu(30)-Arg(31) peptide bond formed in 48 hrs appears to be the result of an equilibrium in the V8-protease catalyzed hydrolytic and synthetic reactions. The reformation of Glu(30)-Arg(31) bond appears to be very selective. V8-protease did not catalyze the peptide bond formation in an equimolar mixture of alpha 31-141 with either alpha 1-23, alpha 1-27, or alpha 28-30 all of which have glutamic acid as the carboxy terminal residue just as in alpha 1-30. The high selectivity in the protease catalyzed formation of Glu(30)-Arg(31) peptide bond suggests that the segment alpha 1-30 of the intact alpha-globin could be exchanged with synthetic analogs of alpha 1-30 using V8-protease without actually isolating the fragment alpha 31-141. Incubation of [14C]-N alpha-DHP-alpha 1-30 with alpha-globin at pH 6.0 and 4 degrees C along with V8-protease in 30% n-propanol indeed resulted in the exchange of the segment alpha 1-30 of alpha-globin with [14C]-N alpha-DHP-alpha 1-30. This V8-protease catalyzed 'segment exchange reaction' should facilitate the preparation of alpha-chains with double mutations from the large number of naturally occurring alpha-chains with single mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

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