Biophys J, July 2000, p. 561-583, Vol. 79, No. 1

Polymerization of Rod-Like Macromolecular Monomers Studied by Stopped-Flow, Multiangle Light Scattering: Set-Up, Data Processing, and Application to Fibrin Formation

Simonetta Bernocco,^* Fabio Ferri,^§ Aldo Profumo, Carla Cuniberti, and Mattia Rocco^*

 ^*Gruppo di Biostrutture and  Servizio di Biologia Molecolare, Istituto Nazionale per la Ricerca sul Cancro, Centro per le Biotecnologie Avanzate, I-16132 Genova,  Dipartimento di Chimica e Chimica Industriale, Università di Genova, I-16146 Genova, and  ^§Dipartimento di Scienze Chimiche, Fisiche e Matematiche, Università dell'Insubria a Como and INFM, I-22100 Como, Italy

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M_w and of the z-average square radius of gyration R_g²_z of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M_w and R_g²_z cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M_w and R_g²_z values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M_w and R_g²_z when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule ~50 nm long. The polymers, grown in the absence of Ca²⁺ by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.

Biophys J, July 2000, p. 561-583, Vol. 79, No. 1
© 2000 by the Biophysical Society   0006-3495/00/07/561/23  $2.00