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Engineering surface charges in a subtilisin: the effects on electrophoretic and ion-exchange behaviour
M R Egmond1, W P Antheunisse, C J van Bemmel
1Unilever Research Laboratorium, Vlaardingen, Netherlands.
Protein Engineering
|June 1, 1994
Summary
Altering enzyme charge affects protein behavior, showing that total charge isn't the sole factor in mobility. Ion type and number of charged residues significantly influence protein adsorption and electrophoretic mobility.
Area of Science:
- Biochemistry
- Protein Engineering
- Physical Chemistry
Background:
- Proteins possess intrinsic electrostatic properties crucial for their function and interactions.
- Genetic engineering allows systematic modification of protein surface charges.
Purpose of the Study:
- To investigate how altering charged amino acid residues impacts the electrostatic properties and behavior of subtilisin Savinase.
- To determine the factors influencing protein electrophoretic mobility and ion-exchange behavior.
Main Methods:
- Genetic engineering to introduce/remove charged residues in subtilisin Savinase.
- Ion-exchange chromatography to assess protein adsorption.
- Electrophoretic mobility measurements in native gels under varying ionic conditions.
Main Results:
- Electrophoretic mobility depends on more than just overall protein charge, especially at low/moderate ionic strengths.
- A linear relationship exists between mobility and the number of charged residues for variants with identical net positive charge.
- The type of salt (e.g., Ca2+, Mg2+, Na+, phosphate) significantly alters ion screening and can even reverse apparent protein charge.
Conclusions:
- Small ions strongly perturb protein charge, with effects dependent on ion type and concentration.
- Protein adsorption is influenced by surface charge density and ion interactions.
- Understanding these electrostatic effects is vital for protein adsorption and surface interaction studies.