Introduction

The visible absorption spectrum associated with the heme protein myoglobin changes as a result of ligand or small molecule binding to the iron center. This spectroscopic handle allows for the experimental determination of the ligand binding or dissociation constant.

The iron atom in the protein has an octahedral coordination. Binding to the four equatorial sites is a substituted porphyrin ring, which is a macrocyclic prosthetic group. A prosthetic group is a non-amino acid portion of a protein. The iron atom is held in the protein through ligation by a histidine residue at an axial site. The remaining axial coordination site is available for oxygen or other ligand binding.

The unsubstituted aromatic porphyrin (C₂₀H₁₄N₄) structure. The two protons inside the ring are lost when the molecule coordinates metal cations in the dianion form. The inner or hole diameter is about 4 Å for the dianion. Many proteins use variously substituted porphyrins as cofactors to coordinate metal ions. The substitution can occur at any of the hydrogen positions on the outside of the ring.

Two views of the myoglobin oxygen storage protein. The amino acid chain of the protein is shown as a strand in both views. The heme group and bound oxygen are shown as space-filling spheres. The color code is: N – blue, O – red, C – grey and Fe – orange. In the right hand view the histidine group that ligates to the iron is shown in a wireframe representation. These views are from a crystal structure found at the protein data bank.

M yoglobin Preparation

Metmyoglobin, the oxidized iron(III) form of myoglobin, is prepared through a dialysis procedure that needs to be started the week before the scheduled laboratory date and attended to during the week. The dialysis tubing needs to be handled carefully. Oils and ions from your hands could contaminate the sample or clog the pores of the tubing. Gloves need to be worn while manipulating the dialysis tubing.

Cut a 9-inch length of Spectrapro-4 dialysis membrane tubing and place it into 150 ml of 0.1 M borate buffer, pH 9.5. The borate buffer is prepared by dissolving sodium borate in water to get a 0.1 M solution, note that although the label say sodium borate the material is really sodium tetraborate. Add sodium hydroxide to raise the pH to 9.5. Cover the beaker with parafilm and store overnight in the refrigerator.

Prepare a 2.0 mM myoglobin (MW 17,800) solution in a 0.1 M borate buffer, pH 9.2. Fill the dialysis tube with the myoglobin solution and secure the open end with a clip. Double check the clip to make sure the solution will not spill out of the dialysis tubing. Put the sealed tubing in a beaker holding 150 ml of a 50 mM potassium ferricyanide (K₃[Fe(CN)₆]) solution, an oxidizing agent. Cover the beaker with parafilm and store overnight in the refrigerator. Store the borate buffer in the refrigerator in order to avoid thermal shock damaging the protein during the dialysis procedure.

Wash the dialysis tubing with buffer and then place the tubing in a beaker containing fresh borate buffer. Cover the beaker with parafilm and store overnight in the refrigerator. Repeat this procedure until no trace of ferricyanide or ferrocyanide is visible in the UV/Visible spectrum of the buffer.

Myoglobin Titration

Prepare 10 ml of 2.0 mM sodium azide solution and 10 ml of 2.0 mM sodium chloride solution using volumetric glassware. It will be difficult to weigh out very small quantities of the salts. It is easier and more accurate to make a more concentrated solution than needed, weighing out a mass large enough to measure easily, and then dilute to get the desired concentration.

Prepare eleven samples with a metmyoglobin concentration of 50 mM and a total volume of 2 ml using a well-plate and a mechanical pipetter. Use the stock 2.0 mM sodium azide (NaN₃) solution in order to have azide concentrations of 0.0, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 and 0.4 mM in your samples. Use the stock 2.0 mM sodium chloride solution to maintain a constant ionic strength of 0.5 mM in all of the solutions. It may be helpful to use EXCEL in order to calculate all of the required volumes. Allow the samples to sit for one hour at room temperature to equilibrate.

Collect an UV/Vis spectrum of one of the samples using the small sample volume quartz cuvette. Wait 5 minutes and collect another spectrum of the previously run sample. If the two spectra of the sample are identical collect spectra for all of the samples.

Data Analysis

Calculate the dissociation constant for the azide-myoglobin complex. The dissociation constant is an equilibrium constant based on the bound myoglobin-azide complex (L-R) in equilibrium with the free myoglobin (receptor, R) and azide (ligand, L).

The more familiar association constant (K) is the reciprocal of the dissociation constant (K_d) calculated from the formula above (K = 1/K_d).

The fraction of the receptor sites that are bound by ligand ([L-R]/[R]_total) is given by the formula for alpha (a). Alpha is found by using the definition of the total receptor concentration and the expression for the dissociation constant and then simplifying to get:

This conveniently provides an expression for alpha (a) as a function only of the free ligand concentration and the dissociation constant. This expression can be used to determine the dissociation constant for the myoglobin-azide complex from spectroscopic data.

Examine the spectra. High quality data for a two-state equilibrium will display an isobestic point. This occurs at a wavelength where the two states have equal absorbance. As a result there is no change in absorbance at that wavelength as the equilibrium is shifted and all of the spectra will overlap at that one wavelength (see below).

Lack of an isobestic point indicates that there is more than a simple two state equilibrium occurring in the sample. Calculate the change in absorbance (DAbs(l)) at the wavelengths where the spectra changes the most, giving a positive and negative peak, by subtracting from the absorbance of the azide containing samples the absorbance of the sample that contains no azide. Graph the two DAbs(l) against the total azide concentrations of the samples. The curve should be increasing at low total azide concentration but should saturate or flatten out at higher total azide concentrations. The value of alpha (a) in the saturated region is 100%.

Calculate DDAbs for each total azide concentration by subtracting one DAbs(l) from the other DAbs(l). Average the DDAbs for the samples in the saturated region in order to get a value for DDAbs_max. The value of alpha (a) for each of the samples that do not have a total azide concentration in the saturated region is the ratio of DDAbs divided by DDAbs_max.

Calculate the value of alpha (a) for each sample that has a total azide concentration not in the saturated region.

Use the value of alpha (a) and the total myoglobin and azide concentrations to calculate the free azide concentration for each sample that has a total azide concentration not in the saturated region.

Use the value of alpha (a) and the free azide concentration to calculate a value for the dissociation constant for each sample that has a total azide concentration not in the saturated region.

A Scatchard plot is a graphical method that can give the dissociation constant from titration data. A graph of the reciprocal of azide-myoglobin concentration (1/[L-R]) against the reciprocal of free azide concentration (1/[L]) will have a slope equal to the dissociation constant divided by the total myoglobin concentration (Kd/[R]_total). The linear relationship between 1/[L-R] and 1/[L] is derivable from the equilibrium expression and the definition for [R]_total.

Calculate the free energy change for azide binding to myoglobin: DG = -RTln(K_a).

Discussion Questions

1) Why is the azide ion (N₃^-) a useful binding probe? How does this ligand compare to O₂?

2) Assign as best you can the nature of the peaks observed in the myoglobin absorption spectrum.

3) What is the dissociation constant and the free energy change of azide binding to myoglobin?

4) What enthalpic and entropic factors contribute to the free energy change of binding? What experiments could be done to measure the enthalpic and entropic contributions to the free energy change?

Useful References

1) Stone, J.R., Sands, R.H., Dunham, W.R., Marletta, M.A.. “Spectral and Ligand Binding Properties of an Unusual Hemoprotein, the Ferric Form of Soluble Guanylate Cyclase”. Biochemistry, 35, 3258, 1996.

2) Marcoline, A.T., Elgren, T.E.. “A Thermodynamic Study of Azide Binding to Myoglobin”. Journal of Chemical Education, 75, 1622, 1998.

3) Milgrom, L.R. The Colors of Life: An Introduction to the Chemistry of Porphyrins and Related Compounds. New York: Oxford University Press, 1997.

4) Marks, G.S.. “Heme and Chlorophyll; Chemical, Biochemical and Medical Aspects”. London: Van Nostrand Co., 1969.

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