Protein estimation by the product of integrated peak area and flow rate

A convenient method for protein estimation is described, making use of uv detectors and peak integrators that are standard equipment on modern high-performance liquid chromatographs to determine the product of integrated peak area and flow rate of eluting protein at 214 nm (AF214). We demonstrate that AF214 is proportional to the amount of eluted protein and describe two approaches for calibrating the integrator, by quantitative amino acid analysis and by determining the elution yield of a known amount of applied protein, allowing direct estimation of protein from AF214. Both approaches yield similar results. The basis for the method is that, for virtually all proteins, absorbance at 214 nm is dominated by the summed contributions from the peptide groups. More accurate estimates can be made when the amino acid composition of the eluting protein is known, since this permits a correction to be made for contributions of amino acid side chains to absorbance at 214 nm. Comparison of AF214 estimates for proteins from the small (30 S) subunit of the Escherichia coli ribosome with those obtained by Bradford analysis shows the latter to give somewhat higher values.