TY - JOUR
T1 - Application of reductive dihydroxypropylation of amino groups of proteins in primary structural studies
T2 - identification of phenylthiohydantoin derivative of ε-dihydroxypropyl-lysine residues by high-performance liquid chromatography
AU - Acharya, A. Seetharama
AU - Sussman, Leslie G.
AU - Manjula, Belur N.
N1 - Funding Information:
This researchw ass upportedi n part by the National Instituteso f Health Grant HL-27183 (to A.S.A.) and a Grant-in-Aid (AHA 83-1102,t o B.N.M.) from the American Heart Associationw ith parts of the funds contributedb y the New York Heart Association.A .S.A. is an EstablishedF ellow of the New York Heart Association, and B.N.M. in an EstablishedIn vestigatoro f the AmericanH eart Association. The interesto f Drs. James M. Manning and Vincent A. Fischetti is gratefully ac-knowledgedT. he assistanceo f Ms. SheenahM . Mische and Ms. Donna Atherton is very much appreciated.
PY - 1984/8/2
Y1 - 1984/8/2
N2 - The general utility of reductive alkylation of amino groups of proteins with glyceraldehyde (2,3-dihydroxypropionaldehyde) in the presence of sodium cyano-borohydride, i.e. dihydroxypropylation, as an aid in generating arginine peptides of proteins by tryptic digestion has been investigated. The dihydroxypropylation of the amino groups of ribonuclease A and the streptococcal Pep M5 protein proceeds predominantly to the stage of monoalkylation. The derivatized lysine namely, ε-dihydroxypropyl-lysine is stable to acid hydrolysis, and is eluted slightly ahead of histidine in the amino acid analyzer. The peptide bonds of ε-dihydroxypropyl-lysine residues are resistant to tryptic digestion. The arginine peptides of dihydroxypropylated ribonuclease A, and dihydroxypropylated streptococcal Pep M5 protein have been isolated by reversed-phase high-performance liquid chromatography (HPLC) of the tryptic digest of the derivatized proteins. The phenylthiohydantoin (PTH) derivative of ε-dihydroxypropyl-lysine has been prepared. It is eluted at a position intermediate to that of the PTH derivatives of proline and tryptophan in reversed-phase HPLC on DuPont Zorbax ODS columns. Thus the PTH-ε-dihydroxypropyl-lysine could be identified during the sequence studies of the dihydroxypropylated peptides. The presence of dihydroxypropyl groups on the ε-amino groups of lysine residues in the dihydroxypropylated peptides does not interfere with the Edman degradation studies. The ease of the dihydroxypropylation reaction, the resistance of the peptide bonds of ε-dihydroxypropyl-lysine residues to trypsin, and the identification of the PTH derivative of ε-dihydroxypropyl-lysine residues by reversed-phase HPLC makes the dihydroxypropylation procedure a valuable addition to the arsenal of procedures for limiting the tryptic digestion to the arginine residues of proteins and peptides.
AB - The general utility of reductive alkylation of amino groups of proteins with glyceraldehyde (2,3-dihydroxypropionaldehyde) in the presence of sodium cyano-borohydride, i.e. dihydroxypropylation, as an aid in generating arginine peptides of proteins by tryptic digestion has been investigated. The dihydroxypropylation of the amino groups of ribonuclease A and the streptococcal Pep M5 protein proceeds predominantly to the stage of monoalkylation. The derivatized lysine namely, ε-dihydroxypropyl-lysine is stable to acid hydrolysis, and is eluted slightly ahead of histidine in the amino acid analyzer. The peptide bonds of ε-dihydroxypropyl-lysine residues are resistant to tryptic digestion. The arginine peptides of dihydroxypropylated ribonuclease A, and dihydroxypropylated streptococcal Pep M5 protein have been isolated by reversed-phase high-performance liquid chromatography (HPLC) of the tryptic digest of the derivatized proteins. The phenylthiohydantoin (PTH) derivative of ε-dihydroxypropyl-lysine has been prepared. It is eluted at a position intermediate to that of the PTH derivatives of proline and tryptophan in reversed-phase HPLC on DuPont Zorbax ODS columns. Thus the PTH-ε-dihydroxypropyl-lysine could be identified during the sequence studies of the dihydroxypropylated peptides. The presence of dihydroxypropyl groups on the ε-amino groups of lysine residues in the dihydroxypropylated peptides does not interfere with the Edman degradation studies. The ease of the dihydroxypropylation reaction, the resistance of the peptide bonds of ε-dihydroxypropyl-lysine residues to trypsin, and the identification of the PTH derivative of ε-dihydroxypropyl-lysine residues by reversed-phase HPLC makes the dihydroxypropylation procedure a valuable addition to the arsenal of procedures for limiting the tryptic digestion to the arginine residues of proteins and peptides.
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U2 - 10.1016/S0021-9673(01)89027-0
DO - 10.1016/S0021-9673(01)89027-0
M3 - Article
C2 - 6436282
AN - SCOPUS:0021754279
SN - 0021-9673
VL - 297
SP - 37
EP - 48
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - C
ER -