Cuantificación y caracterización del comportamiento de partición de la Ribonucleasa A y sus conjugados polímero - proteína en sistemas de dos fases acuosas polímero - sal-Edición Única
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Resumen
Ribonuclease A from bovine pancreas and its PEGylated conjugates has proven to have
several potential therapeutic applications. Aqueous Two-Phase Systems (ATPS) is a
promising primary recovery strategy for the fractionation of proteins and their PEGylated
conjugates. However, in order to characterize the partition behavior of these molecules in
ATPS, an easy to implement method is needed to estimate protein concentration in each
phase. Because RNase A quantification based on colorimetric methods usually renders poor
sensitivity, this paper presents a methodology based on UV absorbance to quantify RNase
A and its PEGylated conjugates on polymer (polyethylene glycol; PEG) and salt (potassium
phosphate; PO4) rich environments, simulating conditions found on polymer – salt ATPS.
The effect of PEG and PO4 concentrations and the effect of grafted PEG chains on RNase A
upon UV absorbance were evaluated. Polymer and salt concentrations have a significant
effect on RNase A absorbance, reflecting the need of specific standard curves in order to
consider the impact of the chemical forming phase upon the extinction coefficient. The
absorbance ratios at 280 nm between mono-PEGylated RNase A/native RNase A and diPEGylated RNase A/native RNase A in PEG and potassium phosphate free environments
were found to be 0.32 and 0.46, respectively, demonstrating that the grafted PEG chains
have also an important effect upon protein light absorbance. The method presented results
in an easy-to-implement alternative, to chromatographic approaches, since just an
UV-visible spectrophotometer or plate reader is required.