School of Biosciences “Global” Hydrodynamic Analysis of the Molecular Flexibility of Konjac Glucomannans Gordon Morris

Outline: 1. Introduction 2. Hydrodynamic characterisations 3. Conformational analyses 4. Conclusions and Future Work

Introduction: Why characterisation of Konjac glucomannan (KGM) is important - Widely used but poorly understood health food supplement - Reported potential interaction with wheat gliadins: application in gluten removal - Biopolymer of interest in diabetes research

KGM: 1. Extracted from the tubers of Amorphophullus Konjac C. Koch 2. Water-soluble gum 3. b-(14) b-D-glucose (G) and b-D-mannose (M) - G:M ratio 1:1.6 - C-6 acetylation (5 – 10 %)

Hydrodynamic characterisations: Sedimentation Velocity in the Analytical Ultracentrifuge - sedimentation coefficient, s020,w - concentration dependence of sedimentation, ks Size Exclusion Chromatography coupled to Multi-Angle Laser Light Scattering - weight average molar mass, Mw Viscometry - intrinsic viscosity, [h]

Results: Sample Mw (g mol-1) [h] (ml g-1) s020,w (S) ks KGM-1 740000 ± 20000 1300 ± 15 3.40 ± 0.02 665 ± 20 KGM-2 695000 1190 ± 25 3.00 ± 0.03 455 KGM-3 305000 ± 10000 775 ± 5 2.50 ± 0.10 275 ± 30 KGM-4 240000 ± 5000 565 ± 10 1.67 ± 0.20 115 KGM-5 210000 475 1.92 160

Conformational analyses: 1. Sedimentation conformation zoning 2. Bushin-Bohdanecky approach 3. Yamakawa-Fujii approach 4. Combined “global” analysis: HYDFIT 5. Mark-Houwink-Kuhn-Sakurada (MHKS) relation 6. Wales-van Holde & frictional ratios

Sedimentation Conformation Zoning: KGM: semi-flexible coil Extra rigid rod e.g. schizophyllan Rigid rod e.g. xanthan KGM: semi-flexible coil Semi-flexible coil e.g. pectin Random coil e.g. pullulan Globular e.g. glycogen Pavlov et al. (1997). Trends in Analytical Chemistry, 16, 401-405.

Bushin-Bohdanecky: Lp ~ 8 nm Semi-flexible coil Bohdanecky (1983). Macromolecules, 16, 1483-1493. Bushin et al., (1981). Vysokomolekulyarnye Soedineniya, A23, 2494-2503.

Yamakawa-Fujii: Lp ~ 33 nm Rigid rod Yamakawa & Fujii (1973). Macromolecules, 6, 407-405.

HYDFIT: Lp ~ 13 nm ML ~ 330 g mol-1 nm-1 Semi-flexible coil Ortega & García de la Torre (2007). Biomacromolecules, 8, 2464-2475.

Summary: Property Value Conformation Zone C Lp (nm) from HYDFIT 13 ± 1 MHKS exponent “a” 0.74 ± 0.01 MHKS exponent “b” 0.32 ± 0.01 ks/[h] 0.4 ± 0.1 f/fo 11 ± 2

Conclusions 1. Discrepancy between Bushin-Bohdanecky and Yamakawa-Fujii approaches - best to use non-biased HYDFIT method 2. Konjac glucomannan has a semi-flexible coil conformation Future Work 1. Characterisation of KGM-gliadin complexes 2. Investigation of KGM-insulin mixtures

Acknowledgements: Prof. Stephen E. Harding & Ali Saber Abdelhameed, University of Nottingham, UK Dr. M. Samil Kök, University of Bolu, Turkey Dr. Jose Garcìa de la Torre & Dr. Alvaro Ortega, University of Murcia, Spain