A biochemist is tasked with measuring the protein concentration in a new biological sample using the alkaline copper solution method. Which principle underlying this method is most crucial for ensuring accurate measurement of protein concentration?

The alkaline copper solution method relies on the fact that copper(II) ions in a strongly alkaline medium form a purple complex with the peptide bonds of proteins. The intensity of this purple color is directly proportional to the number of peptide bonds present, which is in turn proportional to the protein amount. By measuring the absorbance at a specific wavelength and applying Beer‑Lambert’s law, the protein concentration can be calculated accurately. For example, when a sample of bovine serum albumin is added to the alkaline copper solution, the resulting purple color’s absorbance at 650 nm will increase linearly with the protein concentration, allowing precise quantification.