Teaching the use of analytical size exclusion chromatography and light scattering for size determination of challenging proteins

WANDA VALSECCHI; JAVIER SANTOS; JOSÉ M. DELFINO; SANTIAGO E. FARAJ

La Plata, Buenos Aires

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

Analytical size exclusion chromatography (SEC) is a simple yet powerful and widespread method for mass and size estimation of proteins, commonly taught in most biological chemistry undergraduate and graduate courses. However, in an advanced approach to this topic, students are immediately confronted with the fact that SEC results may not accurately describe the molecule under study given that this technique relies on the calibration with reference molecules and assumes certain approximations and ideal conditions?especially protein conformation and absence of interactions with the matrix. Static and dynamic light scattering (SLS and DLS) are two alternative methods that allow, respectively, the absolute determination of the molecular weight and the diffusional coefficient, basically on the basis of the interaction of the molecule with light.In this practical, students make use of SEC, SLS and DLS to determine the molecular weights and hydrodynamic radii of a set of three proteins: bovine serum albumin (BSA), Hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi and human Frataxin (FXN), and analyse the results to make judgments based on the criteria generated during the lecture. These proteins provide a challenging scenario: BSA presents multiple aggregation states, HPRT is found to be a monomer by SEC, but a tetramer by SLS, and FXN is a negatively-charged protein that experience some electrostatic repulsion with the gel matrix.This activity promotes a further understanding of SEC, SLS and DLS, and improves students? skills of constructing meaning from data. The assessment of the implementation showed that students managed to process SEC and SLS data, and gave an explanation to their observations, gaining a deeper awareness of the pros and cons of each technique. As a result, by the end of the activity students were able to correctly select the most appropriate means for the characterization of a sample of a given size, concentration and complexity.