Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics.
Dialysis kinetics measurements have been made to study the effect of ionic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals. Using glutaraldehyde cross-linked dimers of lysozyme, we have determined that both monomers and dimers can escape from 25,000 molecular weight cutoff dialysis membranes with velocity constants of 5.1 x 10(-7) and 1.0 x 10(-7) s(-1) for the monomer and dimer species, respectively. The flux from 25K MWCO membranes has been measured for lysozyme in pH 4.0 buffered solutions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concentrations. Assuming that dimerization is the first step in crystallization, a simple monomer to dimer equilibrium was used to model the flux rates. Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respectively. These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions. At high protein concentrations and high supersaturation, the dimerization model does not describe the data well. However, the Li model that uses a pathway of monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer fits the measured flux data remarkably well suggesting the presence of higher order aggregates in crystallizing solutions.
Department of Chemistry, East Tennessee State University, Johnson City 37614, USA. wilsonl@etsu.-tn.edu
L J
LJ
Wilson
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L
Adcock-Downey
M L
ML
Pusey
D000818:Animals; D002645:Chickens; D003432:Cross-Linking Reagents; D003460:Crystallization; D003956:Dialysis / Q000295:instrumentation / Q000379:methods; D019281:Dimerization; D005260:Female; D005976:Glutaral; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009994:Osmolar Concentration; D012996:Solutions
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D012996:Solutions; D009113:Muramidase; D005976:Glutaral