Branching in High Molar Mass Synthetic Amyloses

Amylose is an occasionally-branched biopolymer and, together with amylopectin, the hyper-branched component, a constituent of starch. Determination of branching in amylopectin on the basis of amyloses may be performed with the help of synthetic amyloses. Synthetic amyloses from enzymatic (phosphorolytic) reaction were checked for their linearity.

Synthetic amyloses were dispersed 1 M KOH (RT, 100mg/ml), diluted with water, filtered at 0,22μm, agitated for 24h under Argon, and ultracentrifuged at 100kxg. The SEC columns were Shodex OHpak HQ806, 805, 804-803, from Showa Denko, Tokyo, Japan, and a DAWN DSP (He-Ne laser, wavelength 632.8nm) and Optilab DSP were used as detectors. Eluant was 0.3 M NaNO₃, pH 10, flow rate 0.5ml/min.

The molar mass averages M (M_w), and root mean square radius R (R_w) were calculated by the Zimm method, RI increment dn/dc=0.146 ml/g. First order fit with respect to M and R. Second virial coefficient A₂ = 0. Slice size 6.25μl. Raw data were imported to Corona 1.40, at least three data sets/sample were averaged.

Average molar mass parameters were assigned using the ASTRA Debye plot option. Averaged raw data were re-imported to Corona. Linear equations for narrow distributed amyloses in the low M range (LMM) calculated to the best fit. Linear extrapolation of the resulting equation in the double logarithmic plot of R vs. M up to 1x10⁸ g/mol was established. Branching calculations [Branching ratio g_M, branches per molecule B and long term branching λ] were performed by the Zimm-Stockmayer equations (trifunctional random branching).

Figure 1

The M_w of LMM synthetic amyloses are narrow, and the branching calculations show strictly linear polymers. HMM amyloses show a broader distribution and a "contraction" of R with increasing M, that indicates that branching increases with increasing M (Fig.1). Branching starts (B=e⁰ ) at approximately 8x10⁵ gmol^-1 (Fig.2). The origin of branching during the enzymatic reaction remains to be investigated. The method has proved to be useful to check branching in HMM biopolymers on the basis of linear LMM biopolymers.

Figure 2

This note graciously submitted by Claudia Niemann, Technical University Berlin, Germany

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