Experiment 9:  SH- and SS- group determination in proteins

Introduction

     When a SH-containing protein of pH 8 is placed into a denaturing solution with 5,5’-dithio-bis(2-nitrobenzoate), aka DTNB, aka Ellman’s reagent, the following reaction occurs:

     The reaction can be tracked by the absorption of thionitrobenzoate at 405 nm. The number of SH- groups in the protein can be calculated using the following: the concentration of ribonuclease A (from beef pancreas, RNase), its molar mass (13 690 Da), the extinction coefficient of thionitrobenzoate (13 600 M^-1·cm^-1), and the measured extinction.

     In order to determine the number of disulfide bridges (S-S groups), we’re going to reduce the SH groups in this experiment with 1,4-dithioerythrite, aka DTE, aka Cleland’s reagent. We’ll then reduce the excess reduction material with gel permeation chromatography before using DTNB to determine the number of SH groups. The difference between this and the theoretical (calculated) number of SH will be used to find the number of disulfide bridges.

Gel permeation chromatography (GPC, gel filtration)

     For this, we’re going to use a stationary phase made up of porous particles. The separation of the sample is based on size. Larger molecules, which can’t fit into the pores on the particles, pass through the fastest; smaller molecules get caught in the pores and thus move through more slowly - thus, the molecules elute according to a decrease in molecular weight. However, since the elution series goes according to diffusion characteristics, this does not apply to molecules of similar structure, eg globular proteins.

Procedure

The following buffer will be required for all solutions:

0.1 M Tris/HCl
1 mM EDTA
0.3% SDS (w/v)
   - pH 8.0

1. Reduction
        A 2 mg/mL ribonuclease solution will be given out by the TAs. Mix 100 µL with 150 µL of a solution of 16.7 mg/mL buffer and incubate 30 minutes at 37°C. For this separation, we’re going use a polypropylene column (1.5 x 5.5 cm) filled with Sephadex G-25 SF. Set up a reservoir (plastic container) containing at least 50 mL mobile phase.

2. Filling the column
        Use a squeeze bottle to fill the column with distilled water to 2-3 cm below the top. (No air bubbles!) Mix the Sephadex G-25 SF with distilled water and add portion-wise until you hit the red mark on the column. Use the water bottle to keep the level up to close to the top. After pouring the gel bed, lay a fret on top parallel to the surface of the gel – no air bubbles! Use the bottle to take off the excess water and equilibrate the column with 5 column volumes of solvent in order to ensure optimal setting of the gel. Carefully remove the fret with a spatula so the differences in ion concentration and hydrostatic pressure of the stationary phase can settle.

3. Calibrating the column
        We’re going to be eluting into a graduated fraction collector. Drain the column till the fluid level sinks to the upper portion of the fret. Apply 200 µL 0.5% dextran blue solution to the gel. If there’s no more solvent above the fret, add 200 µL buffer, which again should be completely absorbed. Add as much buffer as is needed to get the column to run continuously. Take down the volumes at which the elution of the dextran blue starts and ends – the average of these values is the elution volume. Rinse out the column with buffer so we can add our sample.

4. Chromatography of the reduced ribonuclease and determination of SH groups
        Apply 200 µL of the reduced protein solution from a). Collect the eluate in a 10 mL graduated cylinder – collect about 0.1 mL before the dextran blue (protein) starts to come out. Collect the 0.1 mL pre-fraction and the dextran blue volume (protein volume) in a graduated test tube till about 0.1 mL after the calculated end volume is eluted (ie, calculated volume of protein fraction plus 0.1 mL on either end).

        Collect another 5 fractions (0.5 mL each). Top up everything (the blue eluate in whatever volume you calculated earlier and the 5x0.5 mL fractions) up to 3.0 mL. Add DTNB, mix well, let stand 5 minutes, then measure in the photometer at 405 nm (0 = air). The final value should remain constant.

Questions