Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio.
A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.
Tsao
J L
JL
Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854.
Lin
X
X
Lackland
H
H
Tous
G
G
Wu
Y L
YL
Stein
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis* / Q000592:standards; D000621:Aminoisobutyric Acids / Q000032:analysis / Q000592:standards; D002352:Carrier Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D003545:Cysteine / Q000032:analysis; D005022:Ethyldimethylaminopropyl Carbodiimide; D006433:Hemocyanins / Q000737:chemistry; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D012015:Reference Standards
D000596:Amino Acids; D000621:Aminoisobutyric Acids; D002352:Carrier Proteins; D010455:Peptides; C100049:2-aminoisobutyric acid; D010047:Ovalbumin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D003545:Cysteine; D005022:Ethyldimethylaminopropyl Carbodiimide