A Pyrene Excimer Fluorescence (PEF) Study of the Interior of Amylopectin in Dilute Solution

Abstract

The large size of amylopectin represents an experimental challenge for standard analytical techniques to characterize its structure at the molecular level in dilute solution. We introduce a methodology that combines pyrene excimer fluorescence (PEF), the fluorescence blob model (FBM), molecular mechanics optimizations (MMOs), and intrinsic viscosity ([η]) to probe the interior of complex macromolecules with a heterogeneous interior like amylopectin. The combination of PEF, FBM, and MMOs yields the density (ρ fluo ) of nanosized subvolumes of the macromolecule, called blobs within the framework of the FBM, while [η] yields the density (ρ) probed over the length scale of the entire macromolecule. Comparison of ρ and ρ fluo indicates whether matter is either homogeneously (ρ = ρ fluo ) or heterogeneously (ρ < ρ fluo ) distributed inside the macromolecule. The main advantage of the methodology is to shift the focus of the study from the entire polydisperse macromolecule to that of monodisperse blobs where a blob represents the volume probed by a given pyrene derivative. This methodology is thus ideally suited to probe polydisperse heterogeneous macromolecules in solution. It is applied to amylopectin. The finding that ρ was much smaller than ρ fluo for amylopectin in dilute solution led to the conclusion that the side chains of amylopectin are heterogeneously distributed inside the amylopectin interior. The ρ fluo value agreed with the notion that the helical side chains of amylopectin form clusters where the side chains are ∼2.7 nm apart. When ρ and ρ fluo were determined for nanosized amylopectin fragments (NAFs) produced through extrusion, ρ fluo took the same value as for amylopectin while ρ increased with decreasing NAF size, approaching ρ fluo for the smallest NAF. This observation led to the conclusion that the clusters of helical oligosaccharide side chains were loosely connected to each other via linear oligosaccharide segments. These features were captured by the solution-cluster (Sol-CL) model which seems to rationalize many of the experimental observations made about amylopectin.

Interior of Amylopectin and Nano-Sized Amylopectin Fragments Probed by Viscometry, Dynamic Light Scattering, and Pyrene Excimer Fluorescence

“…These concepts were implemented in the present study that provides additional evidence in support of the Sol-CL model introduced earlier [15] and affords a better understanding of the arrangement of oligosaccharide side chains inside NAFs. The experiments described in this paper used the same 21 research-grade NAFs and one amylopectin sample from waxy maize whose characterization has already been reported [15]. In particular, it introduces new experiments, where the degradation of a large NAF by nitric acid was found to yield smaller NAFs, whose D h and [η] matched the values expected of smaller NAFs.…”

“…Twenty-one research-grade NAFs were supplied by EcoSynthetix (Burlington, ON, Canada) by extruding waxy corn starch, which is >99% amylopectin, under different conditions. Their number average particle hydrodynamic diameter (D h ) ranging from 8.3 to 57 nm was measured by DLS [15]. As a precaution to remove possible additives left over during the production of NAFs, all NAFs were purified by precipitation.…”

“…The Solution-Cluster (Sol-CL) model could help in this regard. It was recently introduced to describe the spatial arrangement of the structural motives of amylopectin dispersed in DMSO [15]. It suggests that amylopectin is constituted of dense clusters of oligosaccharide single helices, which are separated from one another by longer linear oligosaccharides.…”

“…While DLS and viscometry probe macromolecules in their entirety over length scales, which ranged from 8.3 to several hundreds of nanometers for the NAFs and amylopectin samples studied, PEF probes macromolecules over a length scale of 4.6 nm or less, when the dye 1- Figure 1. Depiction of the spatial arrangement of the clusters of helices for amylopectin samples of different sizes as predicted by the Solution-Cluster (Sol-CL) model [15].…”

“…These experiments took advantage of the difference in the length scales probed by each technique. While DLS and viscometry probe macromolecules in their entirety over length scales, which ranged from 8.3 to several hundreds of nanometers for the NAFs and amylopectin samples studied, PEF probes macromolecules over a length scale of 4.6 nm or less, when the dye 1-pyrenebutyric acid (PyBA) is used to label polysaccharides at their C2-hydroxyl [15]. Furthermore, the process of excimer formation, where an excimer is the product of the encounter between an excited and a ground-state pyrene, can be quantified by determining the ratio I E /I M of the fluorescence intensity of the excimer (I E ) over that of the monomer (I M ) [16].…”