Quantification of RNase A and Its PEGylated Conjugates on Polymer-Salt Rich Environments Using UV Spectrophotometry

Abstract

Ribonuclease A (RNase A) from bovine pancreas and its PEGylated conjugates has proven to have 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. This paper presents a novel methodology based on UV absorbance to quantify RNase A and its PEGylated conjugates on polymer (polyethylene glycol) and salt (potassium phosphate) rich environments, simulating conditions found on polymer-salt ATPS.

Keywords:

• PEGylated conjugates
• Ribonuclease A
• UV spectrophotometry

Acknowledgments

The authors would like to acknowledge the financial support of CONACyT (Grant 53654) and ITESM Research Chair (Grant CAT161). They would also like to thank Karla Mayolo-Deloisa for her invaluable technical support.

Notes

Systems were designed as described in Materials and Methods section. TLL and compositions for each system were estimated using the binodal curves presented by Zaslavsky (1995).

R² values were obtained by linear regression. The absorption coefficient of RNase A on a 20 mM potassium phosphate pH 7 buffer was found to be 0.5271 with an R² value of 0.9996.