Central European Institute of Technology BRNO I CZECH REPUBLIC Analytical ultracentrifugation Introduction Jan Komárek What is it? analytical ultracentrifugation (AUC) What is it? analytical ultracentrifugation (AUC) What is it? analytical ultracentrifugation (AUC) What is it? analytical ultracentrifugation (AUC) History 1924 - discovery of AUC Svedberg's ultracentrifuge 1926 (Svedberg) Proteins as chemical entities with defined MW 1956 (Chao, Schachman) 1958 (Tissieres, Watson) Structure of ribosomes 1958 (Meselson, Stahl) Semiconservative DNA replication 1963 (Vinograd) DNA supercoiling Instrumentation - speed up to 60 000 rpm - temperature range 0° to 40° C - absorbance optics - wavelength 190 to 800 nm - Rayleigh interference optics - laser wavelength 660 nm An-60 Ti rotor counterbalance Instrumentation (2) Absorbance optical system hi ,._11L. , i ~ i ^ i Hi / 3* i ' i slit movable imaging system xenon flash lamp photomultiplier tube ABS optics -selective detection of absorbing compounds Lambert-Beer Law: (limited linearity) A = C £ I A - absorbance, c- concentration of solute [M], £ -extinction coefficient [IVHcnr1],, /- optical pathlength [cm] 15000 ■ If) B C 10000- 230 nm Xe-lamp emission spectrum 300 400 500 6D0 wavelength [nm] Interference optical system Interference optical system nterference fringes IF optics - sensitive to all components in the solution, including salts! Y = c dn I dc A CCD camera Y- measured fringe displacement [fringes], c - weight concentration [mg-ml-1], dn/dc -refractive index increment, / - optical pathlength, A - wavelength reflective prism Fluorescence detection system (FDS) solid state laser 488 nm photomultiplier tube I pinhole mirror _ cut-off filter (>505 nm) dichroic mirror F = I0Q-£C F - fluorescence intensity, l0 - incident intensity of excitation beam, Q - quantum yield, e - extinction coefficient, c -concentration - higher sensitivity and selectivity - analysis of high-affinity interactions (Kd in the pM range) - analysis of labeled molecules present in complex media (e.g. blood serum, cell lysate) Sedimentation of particles B o CD +■> C (1) Ü (T centrifugal force: Fc may2 r (m - mass of the particle, oj - angular velocity [rad.s-1], r- radial distance) frictional force: Ff = -fu (f- frictional coefficient, u - velocity of particle) Fh = -nin co2 r M buoyant force: mn -mv p- — v p N {m0 - mass of solvent displaced by the particle, v-partial specific volume [cm3-g_1], p - density of solvent, M- molar mass, N- Avogadro's number sedimentation coefficient s: - depends on the mass and shape of particle forces balanced: Fc+Fb+Ff- 0 i Svedberg equation: (definition of sedimentation coefficient) 6.2 6.4 6.6 6.3 7.0 7.2 62 64 6.6 6.8 7JI radius rcmj radius ic.mi Brown, 2008 6.2 64 6.6 6.8 70 radius (cmI M(l-vp) u co2r = s Nf [s] = 1 S (Svedberg, 1013s) Sedimentation of particles B o CD +■> C (1) Ü (T centrifugal force: Fc may2 r (m - mass of the particle, oj - angular velocity [rad.s-1], r- radial distance) frictional force: Ff = -fu (f- frictional coefficient, u - velocity of particle) Fh = -nin co2 r M buoyant force: mn -mv p- — v p N {m0 - mass of solvent displaced by the particle, v-partial specific volume [cm3-g_1], p - density of solvent, M- molar mass, N- Avogadro's number sedimentation coefficient s: - depends on the mass and shape of particle typical values of s: peptides: < 1 S proteins: 1 -10S bacterial ribosome: 70S viruses: 100-600 S forces balanced: Fc+Fb+Ff- 0 i Svedberg equation: (definition of sedimentation coefficient) M(l-vp) u co2r = s Nf [s] = 1 S (Svedberg, 1013s) Sedimentation of particles c 8 CT sedimentation diffusion as a result of random thermal motion of particles First Fick's law: (description of diffusion) JD=-D dc dr {JD- diffusional flux, D- translational diffusional coefficient, dc/dr-concentrational gradient) Stokes-Einstein equation: D = RT limiting case of no diffusion: t = 0 t = 1000 s t = 3000 s Applications What can be studied? What can we learn? purity/homogeneity molar mass size and shape aggregation biomolecular interactions • peptides • proteins, glycoproteins, membrane proteins • nucleic acids (DNA, RNA) • lipids, lipoproteins, liposomes • polysaccharides • viruses, viral vectors • nanoparticles • organic/inorganic polymers Advantages of AUC: • in-solution technique, no interaction with matrix (unlike SEC) • variability in solvent (ionic strength, pH, co-factors) closer to physiological conditions • calibration-free, first-principle method, no imobilization or labeling • non-destructive method • broad dynamic range (small peptides - viruses) • low sample consumption (-hundreds of ug) Sedimentation velocity Sedimentation velocity Sedimentation of a 48 kDa protein (3.4 S) (50,000 rpm, 20 °C, IF detection, 4 hours) Sedimentation velocity meniscus of the sample 1.5-, boundary sedimenting and diffusing in time... radial distance [cm] u)2r Sedimentation velocity Sedimentation velocity Sedimentation velocity meniscus of the sample 1.5-, boundary sedimenting and diffusing in time... 6.4 68 radial distance [cm] cell bottom Lamm equation: (describes the movement of sedimentation boundary in time) dc ~dt D d2c 1 dc + -■ dr r dr dc _ r--h 2c dr Sedimentation velocity raw SV data: 0.6 0.5 0.4 0.3 0.2 0.1 0.0 6.0 . ; //// 6.4 6.6 radius (cm) dt d2c dr2 1 dc +-- r dr - SCO dr fitted data: -0.02 ' / £ // t if f> ft * f,' // f / J /.:'/// ///// +-. 6.2 6.4 6.6 radius (cm) 6.8 result: CO < 2.0 1.5 <2, 1.0 -\ 0.5 - 0.0 Distribution of sedimentation coefficients in the sample Number of peaks corresponds to number of different species in the sample The area under each peak is proportional to the concentration of the species ~i-1———I-1--1-1— 2 4 6 8 10 12 sedimentation coefficient (S) 14 Residual graph showing goodness of the fit Homogeneity Molar mass determination Sedimentation velocity (SV) s -» MW conversion using Svedberg equation: 6 -5 - s->MW 3 4-< 2 - RTs M = --—-(l-vPo)D 1 - 0 - i i i i i i 0 ~2~ 4 ~6 8 10 12 14 sedimentation coefficient (S) Shape frictional ratio f/f0: (describes how much the particle differs in its shape from an ideal sphere) 2/3 (1-VP) f/fo = Ssphere,20,w s20, w ssphere,20w -0.012M' (v- partial specific volume, p - density of solvent, M- molar mass) 8 a) u E S a) ideal sphere ^sphere f/f „ = 1.0 globular protein elongated or glycosylated protein unfolded protein 1.2-1.3 1.5-1.8 >2 Folded or unfolded? Spherical or elongated/flexible? Detection and quantification of aggregates Biopharma industry - mAb as therapeutical drugs Aggregation drug activity immunogenicity pharmacokinetics, pharmacodynamics -» important to detect aggregates during mAb production, formulation and storage monomer (87.4 %) 5 10 15 sedimentation coefficient (S) 20 o "D QJ _N 76 1.0 0.8 0.6 0.4 0.2 0.0 monomer (46.7 %) to $S H—' C CD 1« 2 CM A aW higher oligomers/ small aggregates dimer (15o/o) (2.6 %) ^ J—^-,-,- 0 5 10 15 sedimentation coefficient (S) 20 Sedimentation equilibrium meniscus of the sample 14 4 diffusion sedimentation absorbance [AU] meniscus of the reference bottom of the sector I Reference \ | | | | | | radial distance [cm] exponential shape of curve: one-component system: c( r) = c( r0 )exp(M( 1 - vp)co2(r2 - r02 )/2RT ) c(r) - concentration of molecule at radial distance r, c(r0) - concentration of molecule at reference position r0 3 < c If) time to equilibrium ~ column height2 0.8 0.0 0.01 _W3 03 ■g "I -0.01 PHL, 0.05 mg/ml 0.6 [ 9,500 rpm 11,400 rpm 041" 20,000 rpm 0.2 78.6 kDa 0.00 - M>-(^ft^^^^^ 6.4 6.5 radius (cm) 6.6 Molar mass determination Protein AFL - comparison of SEC and AUC results SIZE-EXCLUSION CHROMATOGRAPHY 1 500 - E 1000 I 500- 1.35 kDa 158 kDa 670 kDa A 44 kü. -1-•-1— 8 10 elution volume [ml] —i-■-1 12 14 by SEC: 30.4 kDa monomer (from amino acid sequence): 34.6 kDa MONOMER! BAD RESULTS! Interaction with matrix ANALYTICAL ULTRACENTRIFUGATION Sedimentation velocity: 3.5 3.0 CO 2.5 < 2.0 1 I 1.5 1.0 0.5 1 0.0 6.0 6.2 6.4 6.6 6.8 7.0 radius (cm) Sedimentation equilibrium: by SE-AUC: 68.0 kDa DIMER! < ^0 ra T3 4.4 S ~ 67.8 kDa 2 4 6 8 10 12 sedimentation coefficient (S) 14 7.0 radius (cm) Overview of AUC techniques SEDIMENTATION VELOCITY (SV) SEDIMENTATION EQUILIBRIUM (SE) • hydrodynamic technique • sensitive to the mass and shape of the particle • performed at high rotor speeds • time: few hours • determination of sediment, coefficient (then s ^ MW) • thermodynamic technique • sensitive to the mass (but not the shape) • performed at lower rotor speeds (several speeds) • time: few days • determination of molar mass directly Velocity Equilibrium Sample Reference Applications: yp Homogeneity ■ Size and shape ■ Molar mass ■ Biomolecular interactions Sample Reference Applications: ■ Molar mass ■ Biomolecular interactions Cole, 1999 Thank you for your attention Sample requirements the requirements dependent on the nature of experiments and a particular sample of interest (some requirements ^negotiable") purity: as pure as possible (>95 % for SE experiment) both sample and solvent necessary - sample should be equilibrated into experimental buffer by dialysis, SEC or spin columns (buffer matching most critical for IF optics) buffers (usually 10-20 mM): should not absorb at wavelength where the sample is measured (e.g. phosphate buffers work well for ABS optics, Tris and Hepes are tolerable at low concentrations for 280 nm) ionic strength (ideally 100-200 mM NaCI): needed to prevent electrostatic interactions (that would affect sedimenation rate and underestimate determined sedimentation coefficient) if possible, substances generating density gradients (glycerol, sucrose, CsCI) should be avoided reductants (DTT, (3ME) should be used at lowest possible concentrations sample concentrations: dependent on absorbtivity and type of the sample, but usually not higheL than 1-2 mg/ml recommended volumes and concentrations for SV/SE experiments (recommended to measure at least 3 different concentrations (to see eventual reversible interactions or sample non-ideality) SV experiment: 450 ul of both sample and reference buffer ^^^^^^i^^^^^^ OD 0.1-1.0 (ABS), loading concentration > 0.1 mg/ml (IF) SE experiment: 150 ul of both sample and reference buffer optimal loading OD 0.1-0.4 (ABS)