Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics

Abstract

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.

Growth of (101) faces of tetragonal lysozyme crystals: determination of the growth mechanism

“…The enthalpies and the pre-exponential constants were determined in our earlier study from data ®ts to the measured (110) growth rates, assuming a dislocation growth model (Li et al, 1995). The results agreed well with the enthalpies estimated from the solubility data (Cacioppo & Pusey, 1991) and estimates of the equilibrium constant for the formation of dimers from aggregation studies (Wilson et al, 1996). These values of the equilibrium constants and enthalpies will be utilized in this study to determine the cluster distributions, as the same distributions can also be assumed to exist in solution for the growth of (101) faces.…”

“…5. Such a mechanism is also suggested by studies showing the presence of clusters in lysozyme solutions under crystallization conditions Behlke & Knespel, 1996;Minezaki et al, 1996;Wilson et al, 1996).…”

Time Dependence of Aggregation in Crystallizing Lysozyme Solutions Probed Using NMR Self-Diffusion Measurements

“…To date, a number of methods have been used to provide information on the earliest stage of the lysozyme crystallization process (i.e., aggregation) including equilibrium ultracentrifugation (Wills et al, 1980), dialysis (Wilson et al, 1996), interferometry (Kim and Myerson, 1994;Albright et al, 1999), light and neutron scattering (Kuehner et al, 1997;Tanaka and Hayakawa, 1999;Georgalis et al, 1999), NMR relaxation dispersion studies (Raeymaekers et al, 1989), and most recently pulsed-gradient spin-echo (PGSE; also known as pulsed field gradient and DOSY) (see Price, 1996Price, , 1997Price, , 1998 NMR diffusion measurements (Price et al, 1999;Price, 2000).…”

The Interface between a Protein Crystal and an Aqueous Solution and Its Effects on Nucleation and Crystal Growth

“…3) values between 1 and 2; thus in these solutions most of the lysozyme molecules are present in small aggregates of about four molecules or in larger, extended, open structures.Experiments with ultracentifugation,48,49 light scattering, 50 neutron scattering, 51-55 and dialysis56 give evidence for the presence of small aggregates of lysozyme molecules in aqueous solutions. There are indications that small aggregates of about four lysozyme molecules act as precursors for the nucleation…”