A simplified method for determination of peptide-protein molar ratios using amino acid analysis

In this report, we have described methods to improve the efficiency of coupling synthetic peptides to keyhole limpet hemocyanin (KLH) and for the analysis of the composition of the resulting peptide-protein conjugates. KLH was first dissolved in buffers containing 3 M guanidine hydrochloride to maintain solubility and derivatized with either of two water soluble, heterobifunctional crosslinkers, m-maleimido-benzoyl-N-hydroxy-sulfosuccinimide ester (SMBS), or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (300:1 molar excess over KLH). Synthetic peptides containing an amino terminal cysteine were then crosslinked to the modified KLH via sulfhydryl reaction with the crosslinker maleimide groups. Following dialysis to remove free peptide, the amino acid composition of the conjugate was determined. The molar ratio of peptide to protein within the conjugate was obtained by comparing the conjugate composition with that of both the KLH and peptide analyzed separately, and by a multiple regression, least squares analysis of the data. This method is generally applicable to the analysis of the molar ratios of protein-protein conjugates of unknown sequence or composition, and requires only the prior determination of the experimental amino acid composition of each component of the conjugate separately.