Abstract
A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.
Highlights
A method for immobilizing protein crystals habseen putational and data acquisition methodologies have greatly devised for determining face growtrhates, and used to reduced the time required for analysis of data only when investigate the growth kineticosf hen egg whitelyso- suitable crystals are available, and advances in biotechnology zyme crystals
A greater understanding of the specific processes involved in protein crystal growth should facilitate growing saturation, with the result thatk = 0.146 % lo-’ cmJs of protein crystals in general
A model of the growthprocess was devel- The most exhaustive single study of protein crystallization oped and the experimental data wuseerdeto determine is that of Schlichtkrull (1956a,1956b,1957a,1957b,1957c, the relativeroles of transport and interfacial kinetics 1957d, and 1957e), who worked with insulin
Summary
A model of the growthprocess was devel- The most exhaustive single study of protein crystallization oped and the experimental data wuseerdeto determine is that of Schlichtkrull (1956a,1956b,1957a,1957b,1957c, the relativeroles of transport and interfacial kinetics 1957d, and 1957e), who worked with insulin. Kam et al (1978), working with lysozyme, used quasi-elastic light scattering to followsize and shape distributions in supersaturated solutions during the prenucleation stage They investigated the concentration gradient about the growing crystals and found a pronounced depletion layer was present. Because ofthe sparse and contradicting literature available, we decided to redetermine the growth rate of lysozyme as afunction of supersaturation and to use the data in conjunction with a diffusoconvective modelfor the growth environment to obtain adetailed understanding of the growth process.Hen egg white lysozyme appears to be a good model because it is readily available in large quantities, is well characterized, and crystallizes readily. $ National Research Council Research Associate at National Aeronautics and Space Administration/Marshall Space Flight Center
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