Microquantification of Proteins by Spectrophotometry. Part II : Application Procedure for Complex Mixture Containing Interfering Substances

In a previous paper we have shown that it was possible to quantify protein solutions at very weak concentrations directly by UV-visible spectroscopy. Nevertheless the protein quantification could not be possible if there is any trace of interferents left in the solution. So it is necessary to eliminate all the interferents to make the measure at 190 and/or 277 nm possible. We have developped a method based on the use of Microcon membranes and centrifugation. Interferents could be eliminated from protein solutions after four centrifugations at 13000 g during 5 min. This procedure allowed the recovering of proteins, with 80 to 99 % yield, and thus making microquantification possible.

This method is particularly interesting for enzymatic solutions after a purification procedure by HPLC where very tiny quantities of protein are recovered. This protocol has been tested on three enzymes; enzymatic activity recovered after four centrifugations was quite high for PEPcase and malic enzyme (71 and 64 % respectively).