Lecture 26: More on Gel Filtration Chromatography and the Trypsin Resurrection Experiment

Transcription

1 Biological Chemistry Laboratory Biology 3515/Chemistry 3515 Spring 2018 Lecture 26: More on Gel Filtration Chromatography and the Trypsin Resurrection Experiment 12 April 2018 c David P. Goldenberg University of Utah goldenberg@biology.utah.edu

2 Gel Filtration Chromatography Also called gel permeation, size exclusion, sizing or molecular sieve chromatography. Beads are made of a porous gel. (similar to gels used for electrophoresis) Separates molecules on basis of size. Larger proteins elute first. (opposite of gel electrophoresis!) Beads have a distribution of pore sizes.

3 Elution Profile for a Gel Filtration Column Absorbance Elution Volume Elution volume is the volume of buffer that flows through the column between when the sample is applied to the top and when a particular protein leaves the column. Can be calibrated with proteins of known size in order to estimate molecular weights of other proteins. Unless a denaturant is present, elution volume usually reflects molecular weight of native protein, with quaternary and tertiary structure intact, unlike SDS gel electrophoresis. Media with different distributions of pore sizes are used to separate molecules of different size ranges.

4 Chromatography as an Equilibrium Process Mobile Phase Stationary Phase Stationary and mobile phases have distinct chemical properties. Molecules partition (thermodynamic equilibrium) between two phases. Molecules that partition preferentially into mobile phase move more rapidly than molecules that prefer the stationary phase. Exchange between the phases has to be rapid, or the molecules will smear out.

5 Quantitative Description of Gel Filtration Chromatography Partition between mobile and stationary phases is determined by K ave, the average fraction of the bead volume that is accessible to the molecule. K ave is a property of the particular molecule and the beads used.

6 K ave Depends on Molecular Size and Distribution of Pore Sizes 1 Fraction of bead volume occupied by matrix Smallest Pores Largest Pores 0 Molecular Size Beads should have a range of pore sizes in order to separate molecules of different sizes.

7 Relationship Between Elution Volume and K ave First, define some volumes: V E =Elution volume, volume of buffer that passes through the column between when the sample is applied and when it leaves the column. V T = Total bed volume V S = Stationary phase volume = volume occupied by beads (including pores) V 0 = Void volume (space between beads) Bed Volume = V T V S

8 Relationship Between Elution Volume and K ave if K ave = 0, molecule is excluded from all pores, V E = V 0 (the space between the beads). if K ave > 0, V E = V 0 + volume of beads that molecule can enter. V E = V 0 + K ave stationary phase volume = V 0 + K ave (V T V 0 ) Rearranged to calculate K ave : K ave = V E V 0 V T V 0

9 Calculation of K ave K ave = V E V 0 V T V 0 V T determined from dimensions of column (V = πr 2 h) V 0 determined as elution volume of molecule much larger than pores. Blue dextran is often used. Elution volume depends on the column dimensions, but K ave should be independent of column size, for a given molecule and type of bead. K ave should lie between 0 and 1, but it might not!

10 Effects of Media and Molecular Size on K ave Superdex peptide Superdex 30 prep grade Superdex 75 Superdex 75 prep grade Superdex 200 Superdex 200 prep grade ,000 Molecular Weight (kda) Media are defined by two properties: Distribution of pore sizes (number) Bead size (grade) Smaller beads give better resolution, but slower flow. For a given pore size distribution, proteins over a range of 10 to 100-fold can be separated. Columns can be calibrated with proteins of known molecular weight and then used to estimate MW of other proteins. Illustration adapted from: Gel Filtration: Principles and Methods, free from GE Healthcare (formerly Amersham Biosciences, formerly Pharmacia) Link to free download:

11 How Does Shape Affect K ave (or Elution Volume)? Cartoon by GE HealthCare:

12 Useful Approximations for Non-spherical Proteins Drawings scaled to represent equal volumes. Axial ratio, c/a, defines asymmetry. Oblate ellipsoid (M&M) Sphere For a given volume ( MW) which is more likely to enter a pore? Prolate ellipsoid (cigar)

13 Clicker Question #1 If the volumes are the same, which will elute first from a gel filtration column? 1 The sphere. Sphere 2 The oblate ellipsoid. 3 They will elute together. Oblate ellipsoid All answers count for now.

14 Clicker Question #2 If the volumes are the same, which will elute first from a gel filtration column? If the volumes are the same, which will elute first from a gel filtration column? 1 The sphere. 2 The prolate ellipsoid. Sphere 3 They will elute together. Prolate ellipsoid All answers count for now.

15 Why Does a Prolate Ellipsoid Elute First? The prolate ellipsoid should be able to enter smaller pores than a sphere of equal volume. But, ellipsoid has to be in the right orientation to enter the smaller pore. Most orientations make it harder to enter a smaller pore. In thermodynamic terms, the ellipsoid has to lose entropy to enter the stationary phase. Elution volume reflects equilibrium between stationary and mobile phase.

16 Effects of Size and Shape on K ave (or Elution Volume) For molecules with the same overall shape, K ave and elution volume decrease with increased molecular weight (over suitable range for a given media). For molecules with different shapes, elution volumes may not reflect molecular weights. When compared to spherical proteins, more asymmetric molecules will appear to have larger molecular weights than they really do. For molecules with a range of shapes, K ave is best correlated with their frictional coefficients and diffusion rates in solution. Stokes radius or hydrodynamic radius, radius of a smooth sphere with the same frictional coefficient. Can be combined with other information (such as sedimentation velocity) to better determine molecular weight and shape.

17 The Last Experiment! Experiment 6, Day 2: Separating trypsin from benzamidine. Is benzamidine inhibition really reversible? 1 Mix trypsin and benzamidine. Save a sample of trypsin without benzamidine. Save a sample of trypsin mixed with benzamidine. 2 Apply mixture to the Sephadex G-25 column. 3 Elute column and collect fractions. 4 Measure A 280 of fractions to identify fractions containing trypsin and benzamidine. 5 Assay trypsin activity in: Uninhibited trypsin Trypsin mixed with benzamidine Trypsin after fractionation (peak fraction)

18 The Last Experiment: Some Considerations To make a meaningful comparison, the trypsin concentrations must be the same in all of the assays. To be detected in the column fractions, the trypsin concentration must be relatively high. 0.1 mg/ml. By comparison, the trypsin samples used in our previous rate measurements had concentrations of mg/ml. If we dilute the trypsin samples in this experiment to make the rate slow enough to measure, we will also dilute any inhibitor present. The solution: Use a substrate that is cleaved more slowly than Bz-Phe-Val-Arg-p-NA.

19 Two Chromogenic Substrates for Trypsin Bz-Phe-Val-Arg-p-nitroanilide Bz-Arg-p-nitroanilide

20 Models of the Two Substrates Bound to Trypsin Bz-Phe-Val-Arg-p-nitroanilide Bz-Arg-p-nitroanilide How will Km and kcat for the two substrates differ?

21 Clicker Question #3 For Bz-Arg-p-nitroanilide, compared to Bz-Phe-Val-Arg-p-nitroanilide, 1 K m will be greater, and k cat will be the same. 2 k cat will be smaller, and K m will be the same. 3 K m will be greater, and k cat will be smaller. 4 K m will be greater, and k cat will be greater. 5 K m will be smaller, and k cat will be smaller. All answers count for now.