SEC MALLs and AUC. 2. Conformation and flexibility Viscometry,

Transcription

1 1. Molecular weight distribution analysis SEC MALLs and AUC 2. Conformation and flexibility Viscometry, AUC, Light scattering

2 Lecture 4. Analytical l Ultracentrifugation t ti I: Molecular weight and conformation Steve Harding

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5 Molecular weight: analytical ultracentrifugation

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9 1. Molecular weight and molecular weight distribution ib ti analysis 2. Conformation and flexibility analysis - general (rods, spheres, coils etc) - polymer flexibility - protein conformation: ellipsoids and bead models

10 Analytical ultracentrifugaton: Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Sedimentation Equilibrium Centrifugal force Diffusion Solution conc, c Rate of movement of boundary sed. coeff distance, r conc, c distance, r STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS

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18 Extraction of M w,app from sedimentation equilibrium and MSTAR analysis Chitosan G213 M w,app cell bottom

19 Extraction of M w,app from sedimentation equilibrium and MSTAR analysis xanthan M w = ( )x10 6 g/mol

20 SEC - sedimentation equilibrium mol. wt distribution: alginate Ball A, Harding SE & Mitchell J, Int. J. Biol. Macromol., 1988

21 Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Sedimentation Equilibrium Centrifugal force Diffusion Solution conc, c Rate of movement of boundary sed. coeff distance, r conc, c distance, r STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS

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26 Guar, 0.75 mg/ml Sedimentation g(s*) plot : f l i f th h from analysis of the change with time of the whole concentration profile

27 Sedimentation velocity g*(s) plot: starch Tester R, Patel T, Harding, S. Carbohydrate Research, 2006

28 Multi-Gaussian fit estimates proportions p of each species too:

29 Converting a sedimentation coefficient distribution to a molecular weight distribution s 20 w ~ M b 20,w Harding, S. Adv. Carb. Chem. Biochem, 1989

30 Converting a sedimentation coefficient distribution to a molecular weight distribution Molecular weight distribution no column or membrane needed s 20,w ~ M b (b=0.5) Harding, S. Adv. Carb. Chem. Biochem, 1989

31 Converting a sedimentation coefficient distribution to a molecular weight distribution Glycoconjugate vaccine too large for SEC-MALLs analysis s 20,w ~ M b Harding, S., Abdelhameed, A., Morris, G. (2010)

32 1. Molecular weight and molecular weight distribution ib ti analysis 2. Conformation and flexibility analysis - general (rods, spheres, coils etc) - polymer flexibility - protein conformation: ellipsoids and bead models

33 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 ) 1 ls-g(s) (f fringes s Sedimentation coefficient (Svedberg)

34 s o 20,w and k s extraction x10 12 s 0 8.0x ,b = 2.04 (0.07) S k s = 270 (25) mlg x10 12 Svedberg -1 ) sec -1 1/s (S 1/s 20,w 7.0x x x x10 12 slope=k s /s o 20,w 5.0x /s o 20,w 45x10 4.5x x x x x x10-3 Concentration (gml -1 ) Concentration g/ml

35 General conformation analysis: the Haug Triangle

36 Power law, Scaling or MHKS relations: Sphere Rod Coil [η] ~ M 0 [η] ~ M 1.8 [η] ~ M s o 20 w ~ M 0.67 s o 20 w ~ M 0.15 s o 20 w~ M ,w 20,w 20,w R g ~ M 0.33 R g ~ M 1.0 R g ~ M g g g

37 Mark-Houwink-Kuhn-Sakurada Power law plot Galactomannans a=

38 Change in Conformation Rollings, 1992

39 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

40 Conformation Zoning: 3.5 log (10-11 k s M L ) A B C D E Pavlov, Rowe & Harding, Trends in Analytical Chemistry,1997 log (10 12 [s]/m L )

41 3.5 Bovine glycogen A B Pectins Pullulans log (10-11 k s M L ) C D E log (10 12 [s]/m L )

42 Worm-like Chain Flexibility parameter: Persistence length L p Contour Length Kuhn-statistical length λ -1 = 2L p

43 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

44 Bushin-Bohdanecky relation [ ] 2 1/ 2 1/ 3 1/ 0 3 1/ 0 3 1/ 2 2 w p L w M M L B M A M Φ + Φ = η[ ] L M η Yamakawa-Fujii relation ( ) = / / 0 0 w w L L M M A A L M M N v M s πη ρ p L p L A L M L M N πη

45 Global Hydfit plot: xyloglucan (g. mol -1. nm -1 ) M L L p (nm) Patel et al, Carbohydrate Polymers, 2007

46 Flexibilities of carbohydrate polymers b h d t l Carbohydrate bhd Pl Polymer L p (nm) Pullulan Amylose 2.8 Pectin (69% esterified) Pectin (0% esterified) 34 DNA 45 Schizophyllan py Scleroglucan Xanthan 210

47 Protein conformation: ellipsoids and beads Software tti h h Ellips1 (ellipsoids of revolution) Ellips2, Ellips3, Ellips4 (general ellipsoids) Software es/macromol Hydro, Solpro, HydroPro

48 Ellipsoid axial ratio determinations wheat protein gliadins Structure and heterogeneity of gliadin: a hydrodynamic evaluation S. Ang et al, Eur. Biophys. J. (2009)

49 Ellipsoid axial ratio determinations wheat protein gliadins Structure and heterogeneity of gliadin: a hydrodynamic evaluation S. Ang et al, Eur. Biophys. J. (2009) ELLIPS1 h

50 Demonstration of ELLIPS1 & ELLIPS2 programs: download from For a wide variety of hydrodynamic parameters including ν (from intrinsic viscosity) see Lecture 1 notes or P (from sedimentation or diffusion measurements) see Lecture 3 notes: ν = [η] /v s P = (f/f o o) ). (v/v s s) 1/3 where (f/f o ) = (k B T/6πη o ){(4πN A /3vM) 1/3 }/D o 20,w = (M(1-vρ 1/3 o o )/N A 6πη o ){(4πN A /3vM) }/s 20,w

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52 For more complicated shapes: BEAD & SHELL MODELS Hydro, Solpro, HydroPro etc IgE IgG1

53 Follow up bibliography: 1. Serydyuk, I.N., Zaccai, N.R. and Zaccai, J. (2006) Methods in Molecular Biophysics, Cambridge, Chapters D1 and D4 2. Harding, S.E., Rowe, A.J. and Horton, J.C., Eds (1992) Analytical Ultracentrifugation in Biochemistry and Polymer Science, Royal Soc. Chem. Cambridge 3. Scott, D.J. et al (2005) Analytical Ultracentrifugation. Techniques and Methods, Royal Soc. Chem. Cambridge 4. Harding, S.E. & Johnson, P.J. (1985) The concentration dependence d of macromolecular l parameters, Biochem. J. 231, Harding, S.E. (1995) On the hydrodynamic analysis of macromolecular conformation. Biophys Chem 55, Garcia de la Torre et al (1997) SOLPRO: theory and computer ( ) y p program for the prediction of SOLution PROperties of rigid macromolecules and bioparticles. Eur. Biophys. J. 25,