Abstract
Affinity coelectrophoresis (ACE) was developed as a tool to measure the strengths of interaction between proteoglycans (PGs) or glycosaminoglycans (GAGs) and proteins, and to assess the specificity of the interaction (i.e., to detect and fractionate GAG or PG sample constituents that differentially bind to protein) (1). In ACE, trace concentrations of radiolabeled GAG or PG are subjected to electrophoresis through agarose lanes containing protein at various concentrations. The electrophoretic pattern of the radiolabeled GAG or PG is then visualized by autoradiography, or using a phosphorimager, and the apparent dissociation constant (K d ) is calculated as the protein concentration at which the GAG or PG is half-shifted from being fully mobile at very low protein concentrations (or between protein-containing lanes) to being maximally retarded at saturating protein concentrations (see Figs. 1–Figs. 3).
Analytical ACE schematic. Top panel: ACE gels poured using a casting stand and Teflon combs and strips as shown in Fig. 5 are used to create nine parallel rectangular wells, which are filled with protein-agarose mixtures, each at a different protein concentration. Radiolabeled GAG or PG is loaded into the slot above the protein-containing wells (shown as a dark line), and after electrophoresis of the GAG or PG through the protein-containing lanes, its migration as a function of protein concentration is visualized by autoradiography or phosphorimaging (shown here as a pattern of peaks and valleys). The degree of GAG/PG retardation at the various protein concentrations is used to calculate the apparent K d of GAG- or PG-protein binding (see text for details). Artwork by Shawn M. Sweeney.
(opposite page) ACE analysis can reveal the affinity of interactions between PGs or GAGs and various proteins. For these experiments, syndecan-1 was electrophoresed through types I–VI collagens in ACE gels. (A) Images of PG migration patterns were obtained using a phosphorimager. The electrophoretograms indicate that some collagens bind strongly to syndecan-1 (e.g., type V), and others bind weakly (e.g., type II). Protein concentrations in nM are shown beneath gels. (B) Calculation of affinities of syndecan-1 for various human collagens. From each electrophoretogram in panel (A), retardation coefficients (R) for syndecan-1 were determined (see text) and are plotted against protein concentration. Smooth curves represent nonlinear least-squares fits to the equation R = R∞ (1 + (K d /[protein])2). Data are adapted from (5).
ACE analysis can reveal selectivity in PG- or GAG-protein interactions. Example of ACE analysis of the interactions between a basic peptide and 35S-sulfate metabolically labeled PGs/GAGs secreted by endothelial cells in vitro. ACE gel image was obtained using a phosphorimager. At least two populations of PG/GAG, seen as two bands of radiolabeled material migrating with different mobilities at low protein concentrations (<50 nM), indicates heterogeneity in size and/or charge density within the PG/GAG mixture. Potential heterogeneity in PG/GAG-peptide interactions is also obvious at a peptide concentration of 250 nM, in which a fractionation of the PG species through the peptide-containing lane is evident as a broad smear throughout the lane, and as a sharp band that migrates approximately halfway down the lane. Thus, components of the PG/GAG sample are binding strongly to the peptide (i.e., are retained closer to the top of the peptide-containing lane), and others are binding more weakly to the peptide (i.e., are not significantly retained and migrate further within the peptide-containing lane). In such cases preparative ACE can be used to recover differentially binding PG/GAG populations for further characterization. Data are adapted from (11)
Preparative ACE schematic. Top panel: A preparative ACE gel is poured using a casting stand as shown in Fig. 5 , except instead of using protein well-forming Teflon combs, a single Plexiglas block is used to create one large rectangular well to be filled with a single protein-agarose mixture. Radiolabeled GAG or PG is loaded into the slot above the proteincontaining wells (shown as a dark line to the left in the gel schematic), and electrophoresed through the protein-containing zone. Middle panel: The agarose gel surrounding the protein-containing zone is trimmed away, and the remaining protein-agarose block is sectioned into 2-mm-thick segments. The amount of radiolabeled GAG or PG in each segment is then determined. Bottom panel: Actual plot of CPM/fraction of heparin octasaccharide mixture electrophoresed through 1000 nM type I collagen, showing the partial resolution of four differentially binding populations. (From San Antonio and Lander, unpublished data). Artwork by Drew Likens.
Oblique view of apparatus for pouring two ACE gels, each with protein-containing lanes 15 mm in length. Plexiglas casting stand contains a clear piece of gel bond (not visible in this photograph), on which are placed two Teflon combs that are each used to create 9 agarose-protein containing lanes, and two Teflon strips that are each used to create a GAG/PG loading slot. The stand is bordered on two sides by masking tape, which retains the agarose and Teflon strips in place. After filling the stand with agarose, upon solidification the combs and strips are removed, forming two ACE gel templates to be run as described in the text and shown diagramatically in Fig. 1 .
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© 2001 Humana Press Inc., Totowa, NJ
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San Antonio, J.D., Lander, A.D. (2001). Affinity Coelectrophoresis of Proteoglycan-Protein Complexes. In: Iozzo, R.V. (eds) Proteoglycan Protocols. Methods in Molecular Biology™, vol 171. Humana Press. https://doi.org/10.1385/1-59259-209-0:401
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DOI: https://doi.org/10.1385/1-59259-209-0:401
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