Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I-Structural and functional alterations

Understanding the chemistry of protein modification by formaldehyde is central to developing improved methods to recover proteins from formalin-fixed paraffin-embedded tissues for proteomic analysis and to improve protein immunoreactivity for immunohistochemical studies. We used biophysical techniques to investigate the effects of formaldehyde treatment on bovine pancreatic ribonuclease A (RNase A). Treatment of RNase A with formaldehyde was shown by gel electrophoresis to lead to the rapid formation of intra- and intermolecular protein cross-links. Thermal studies revealed that these protein cross-links significantly increased the thermal denaturation temperature of RNase A preparations. Analysis of formaldehyde-treated RNase A oligomers isolated by gel chromatography revealed that intramolecular protein cross-links are primarily responsible for the increase in protein thermostability. Formaldehyde treatment also lowered the isoelectric point of the enzyme from 9.45 to the 6.0-7.4 range. Optical spectroscopic studies demonstrated that the formaldehyde-induced modifications did not significantly alter the secondary or tertiary structure of RNase A. Heating formaldehyde-treated RNase A at 65degreesC resulted in a significant reversal of the protein intra- and intermolecular cross-links and led to a partial restoration of enzymatic activity.