title: Determination of protein concentration for protein-protein conjugates using ultraviolet absorption.
abstract: The present study reports a method to determine the total protein concentration or concentration of a protein of interest in a protein-protein conjugate using ultraviolet absorption, after determining the molar ratio of proteins in the conjugates, from which an extinction coefficient can be calculated. A Microsoft Excel solver-based template using amino acid analysis data was developed for determining the molar ratio. The percent mass of each protein in the conjugate is calculated from the amino acid composition data using the least squares method in the Microsoft Excel solver function, and the percent mass is converted to molar portion of each protein using corresponding molecular weight. A molar ratio is obtained by dividing the molar portion of protein 1 by the molar portion of protein 2. A weighted extinction coefficient is calculated using the molar ratio, and the total protein concentration is determined using ultraviolet absorption at 280 nm. The accuracy of the method was verified using mixtures of known proteins. The present study provides a rapid, simple and accurate method for determining protein concentration in protein-protein conjugates.
authors.affiliation: Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. dzhu@niaid.nih.gov
authors.forename: Daming
authors.initials: D
authors.lastname: Zhu
authors.forename: Feng
authors.initials: F
authors.lastname: Qian
authors.forename: Yimin
authors.initials: Y
authors.lastname: Wu
authors.forename: David S
authors.initials: DS
authors.lastname: Jones
authors.forename: Christopher
authors.initials: C
authors.lastname: Rowe
authors.forename: David L
authors.initials: DL
authors.lastname: Narum
authors.forename: Patrick
authors.initials: P
authors.lastname: Duffy
authors.forename: Louis H
authors.initials: LH
authors.lastname: Miller
authors.forename: Allan
authors.initials: A
authors.lastname: Saul
mesh_terms: D000465:Algorithms; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods; D006868:Hydrolysis; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012984:Software; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: D000596:Amino Acids; D010455:Peptides; D011506:Proteins