Amino acids protein elution-concentration

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side chains of the amino acids tyrosine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their content of tyrosine and tryptophan vary to any significant extent. Hence, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process. 

Analysis of the protein content of active fractions revealed two bands in the fraction eluted by urea (Fig, 1 A). The apparent molecular masses of the proteins, 90 and 70 kDa, corresponded well to the molecular masses calculated from the deduced amino acid sequences of GyrA (90,460 kDa) and GyrB (72,459 Da). The concentration of DNA gyrase (GyrA and GyrB) in fractions was estimated to be 40 ng/jjtl, i.e., about 120 nM with a calculated molecular mass of 325,838 Da for the heterotetramer A2B2. 

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