Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: a case study with lysozyme.
Lysozyme-lysozyme interactions were assessed in the native state at 25 degrees C as well in the denatured state at 80 degrees C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.
Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany.
Virginie
V
Le Brun
Wolfgang
W
Friess
Stefan
S
Bassarab
Patrick
P
Garidel
D002846:Chromatography, Affinity / Q000379:methods*; D003102:Colloids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009113:Muramidase / Q000737:chemistry*; D009997:Osmotic Pressure; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D012965:Sodium Chloride / Q000737:chemistry*; D055672:Static Electricity; D013314:Stress, Mechanical; D013696:Temperature
D003102:Colloids; D012965:Sodium Chloride; D009113:Muramidase