Hydrodynamic Characterisation
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1 Hydrodynamic Characterisation Viscometry SEC-MALLs Analytical Ultracentrifugation Stephen Harding, NCMH University of Nottingham
2 NCMH at Nottingham: An International Facility for characterising sizes/shapes and interactions of macromolecules in solution
3 Hydrodynamic characterisation 1. Viscosity 2. Heterogeneity, Molecular weight & distribution 3. Conformation in solution 1: Viscometry. 2: SEC-MALLs & analytical ultracentrifugation. 3: Viscometry, SEC-MALLs & analytical ultracentrifugation.
4 1. Viscosity by precision viscometry
5 Viscosity by Precision viscometry [η] Intrinsic viscosity, ml/g
6 Types of Viscometer: 1. U-tube (Ostwald or Ubbelohde) Ostwald Viscometer
7 Types of Viscometer: 1. U-tube (Ostwald or Ubbelohde) Extended Ostwald Viscometer
8 Auto-timer Coolant system Density meter Solution Water bath o C
9 Types of Viscometer: 2. Cone & Plate (Couette) Couette-type Viscometer
10 3. Rolling ball capillary viscometer
11 Definition of viscosity: For normal (Newtonian) flow behaviour: viscosity τ = (F/A) = η. (dv/dy) shear rate shear stress η = τ/(dv/dy) units: (dyn/cm 2 )/sec -1 At 20.0 o C, η(water) ~ 0.01P = dyn.sec.cm -2.. = POISE (P)
12 Viscosity of biomolecular solutions: A dissolved macromolecule will INCREASE the viscosity of a solution because it disrupts the streamlines of the flow:
13 We define the relative viscosity η r as the ratio of the viscosity of the solution containing the macromolecule, η, to that of the pure solvent in the absence of macromolecule, η o : η r = η/η o no units For a U-tube viscometer, η r = (t/t o ). (ρ/ρ o )
14 Reduced viscosity The relative viscosity depends (at a given temp.) on the concentration of macromolecule, the shape of the macromolecule & the volume it occupies. If we are going to use viscosity to infer on the shape and volume of the macromolecule we need to eliminate the concentration contribution. The first step is to define the reduced viscosity η red = (η r 1)/c If c is in g/ml, units of η red are ml/g
15 The Intrinsic Viscosity [η] The next step is to eliminate non-ideality effects deriving from exclusion volume, backflow and charge effects. We measure η red at a series of concentrations and extrapolate to zero concentration: [η] = Lim c 0 (η red) polysaccharide η red 200 units: ml/g (ml/g) 100 protein c (g/ml)
16 Concentration Extrapolation 2 main forms Huggins equation: η red = 1+ [ η] ( [ η] c) K H Kraemer equation: η inh [ η] ( [ η] c) = 1 K K 500 Viscosity, η red, η inh, [η ], ml/g Concentration, g/ml Huggins Kraemer
17 Another important relation is the SOLOMON-CIUTÂ approximation, which permits the approximate evaluation of [η] without a concentration extrapolation. [ η] [2( η rel 1) 2ln c nre l 1 ] Viscosity, η red, η inh, [η ], ml/g Concentration, g/ml Solomon-Ciutâ
18 M w (g/mol) [η] (ml/g) Glucose Myoglobin Ovalbumin Haemoglobin Tomato bushy stunt virus Fibrinogen Myosin Chitosan Chitosan GLOBULAR, SPHERES COILS, RODS
19 2. Heterogeneity & molecular weight: SEC-MALLs
20 Molecular Weight: Light scattering MALLs detector
21 Molecular Weight: Light scattering photodiode detectors incident beam transmitted beam
22 Molecular Weight: Zimm plot 10 6.(Kc/R θ ) 1/M w R g from slope sin 2 (θ/2) + kc (k=100)
23 Molecular Weight: SEC MALLS
24 Molecular Weight: SEC MALLS Colonic mucin Fogg FJJ et al, Biochemical Journal.1996
25
26 Can also couple a special type of viscometer on-line Differential pressure viscometer
27
28
29
30 Analytical Ultracentrifugation
31 Optima XLA/XLI
32
33 2 types of AUC Experiment: Sedimentation Velocity Sedimentation Equilibrium Centrifugal force Top view, sector of centrifuge cell Air Solvent Centrifugal force Diffusion Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w 1S=10-13 sec conc, c distance, r STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS
34 Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w Sedimentation coefficient, S
35 Chitosan G213, 0.5 mg/ml meniscus Cell base
36 Chitosan G213, 0.5 mg/ml Sedimentation velocity g*(s) plot from analysis of the change with time of the whole concentration profile ls-g(s) (fringes Svedberg -1 ) s* (Svedberg)
37 Chitosan (3.2 mg/ml) + HA (0.8 mg/ml) ls-g(s) s*
38 Sedimentation velocity g*(s) plot: starch Tester R, Patel T, Harding, S. Carbohydrate Research, 2006
39 2 types of AUC Experiment: Sedimentation Velocity Sedimentation Equilibrium Centrifugal force Top view, sector of centrifuge cell Air Solvent Centrifugal force Diffusion Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w 1S=10-13 sec conc, c distance, r STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS
40 M* analysis of sedimentation equilibrium Creeth JM & Harding SE J. Biochem. Biophys. Meth., 1982
41 Extraction of M w,app from sedimentation equilibrium and MSTAR analysis Chitosan G213 M w,app cell bottom
42 Extraction of M w,app from sedimentation equilibrium and MSTAR analysis xanthan M w = ( )x10 6 g/mol
43 SEC - sedimentation equilibrium mol. wt distribution: alginate Ball A, Harding SE & Mitchell J, Int. J. Biol. Macromol., 1988
44 3. Conformation in solution
45 Conformation in solution 1. Experimental data required Molecular weight: SEC-MALLs reinforced by sed. equilibrium [η], s, R g 2. Modelling strategies General conformation type (rod, coil or sphere etc.) Asymmetry, hydration, branching and flexibility Persistence length
46 Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w + concentration dependence parameter k s
47 Citrus pectin mg/ml s = 1.21 S 2.04 mg/ml s = 1.36 S 1.40 mg/ml s = 1.49 S 1.13 mg/ml s = 1.56 S 0.79 mg/ml s = 1.61 S 0.23 mg/ml s = 1.99 S ls-g(s) (fringes s -1 ) Sedimentation coefficient (Svedberg)
48 s o 20,w and k s extraction 8.5x10 12 s 0 8.0x ,b = 2.04 (0.07) S k s = 270 (25) mlg x /s (Svedberg -1 ) 1/s 20,w sec x x x x10 12 slope=k s /s o 20,w 5.0x x /s o 20,w x x x x x10-3 Concentration (gml -1 ) Concentration g/ml
49 Molecular Weight: Zimm plot 10 6.(Kc/R θ ) 1/M w R g from slope sin 2 (θ/2) + kc (k=100)
50 Haug Triangle
51 Sphere [η] ~ M 0 Rod [η] ~ M 1.8 Coil [η] ~ M s o 20,w ~ M0.67 R g ~ M 0.33 s o 20,w ~ M0.15 R g ~ M 1.0 s o 20,w ~ M R g ~ M
52 Mark-Houwink-Kuhn-Sakurada Power law plot Galactomannans a=
53 Change in Conformation Rollings, J in Laser Light Scattering in Biochemistry (Harding, Sattelle and Bloomfield eds). 1992
54 Sphere [η] ~ M 0 Rod [η] ~ M 1.8 Coil [η] ~ M s o 20,w ~ M0.67 R g ~ M 0.33 s o 20,w ~ M0.15 R g ~ M 1.0 s o 20,w ~ M R g ~ M k s /[η] ~1.6 k s /[η] <1 k s /[η] ~1.6
55 Conformation Zoning: Zone A: Extra-rigid rod: schizophyllan Zone B: Rigid Rod: xanthan Zone C: Semi-flexible coil: pectin Zone D: Random coil: dextran, pullulan Zone E: Highly branched: amylopectin, glycogen
56 Conformation Zoning: 3.5 log (10-11 k s M L ) A B C D E Pavlov et al. Trends in Analytical Chemistry,1997 log (10 12 [s]/m L )
57 3.5 Bovine glycogen A B Pectins Pullulans 2.0 log (10-11 k s M L ) C D E log (10 12 [s]/m L )
58 Worm-like Chain Flexibility parameter: Persistence length L p Contour Length Kuhn-statistical length λ -1 = 2L p
59 Worm-like Chain Flexibility parameter: Persistence length L p Theoretical limits: Random coil L p = 0 Rigid rod L p = infinity Practical limits: Random coil L p ~ 1-2nm Rigid rod L p ~ 200nm
60 [ ] 2 1/ 2 1/ 3 1/ 0 3 1/ 0 3 1/ 2 2 w L p L w M M L B M A M Φ + Φ = η ( ) = / / p L w p L w A L L M M A A L M M N v M s πη ρ Bushin-Bohdanecky relation Yamakawa-Fujii relation
61 800 Global plot: xyloglucan 700 M L (g. mol -1. nm -1 ) L p (nm) Patel et al, Carbohydrate Polymers, 2007
62 Flexibilities of carbohydrate polymers Carbohydrate Polymer Pullulan Heparin Amylose Cellulose Pectin (69% esterified) Pectin (0% esterified) DNA Schizophyllan Scleroglucan Xanthan L p (nm)
63 Next time: how we can use hydrodynamics to study polysaccharide interactions
64 Follow up bibliography: Outline introduction to the techniques: Tombs, M.P. and Harding S.E. (1997) An Introduction to Polysaccharide Biotechnology Taylor & Francis, London Chapter 1 Viscometry: Harding, S.E. (1997). The Intrinsic Viscosity of Biological Macromolecules. Progress in Measurement, Interpretation and Application to Structure in Dilute Solution. Progress in Biophysics and Molecular Biology (T.L. Blundell ed.) 68, Light Scattering: Harding, S.E., Sattelle, D.B. & Bloomfield, VA. Eds (1992) Laser Light Scattering in Biochemistry Royal Soc. Chem. Cambridge Chapters by Wyatt and Rollings Analytical ultracentrifugation: Harding, S.E. (2005) Analysis of polysaccharides by ultracentrifugation: size, conformation and interactions in solution. in Advances in Polymer Science (Polysaccharides I: Structure, Characterisation and Use) Ed. T. Heinze, 186, Chap. 5.
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