Turbidity as a probe of tubulin polymerization kinetics: a theoretical and experimental re-examination.
We report here an examination of the validity of the experimental practice of using solution turbidity to study the polymerization kinetics of microtubule formation. The investigative approach proceeds via numerical solution of model rate equations to yield the time dependence of each microtubule species, followed by the calculation of the time- and wavelength-dependent turbidity generated by the calculated distribution of rod lengths. The wavelength dependence of the turbidity along the time course is analyzed to search for generalized kinetic regimes that satisfy a constant proportionality relationship between the observed turbidity and the weight concentration of polymerized tubulin. An empirical analysis, which permits valid interpretation of turbidity data for distributions of microtubules that are not long relative to the wavelength of incident light, is proposed. The basic correctness of the simulation work is shown by the analysis of the experimental time dependence of the turbidity wavelength exponent for microtubule formation in taxol-supplemented 0.1 M Pipes buffer (1 mM GTP, 1 mM EGTA, 1 mM MgSO4, pH 6.4). We believe that the general findings and principles outlined here are applicable to studies of other fibril-forming systems that use turbidity as a marker of polymerization progress.
Hall
Damien
D
Department of Chemistry, Cambridge University, Cambridge CB2 1EW, UK. drh32@cam.ac.uk
Minton
Allen P
AP
D000818:Animals; D001923:Brain Chemistry; D002021:Buffers; D002417:Cattle; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D009391:Nephelometry and Turbidimetry; D017239:Paclitaxel / Q000494:pharmacology; D011108:Polymers; D013053:Spectrophotometry; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure
D002021:Buffers; D011108:Polymers; D014404:Tubulin; D017239:Paclitaxel