SPY: A Spectrophotometric Study of Hemoglobin

Summary

Three different, physiologically relevant redox states of hemoglobin will be studied using UV-visible spectroscopy. The molar absorptivity at λmax for the Soret band (380-430 nm) will be determined by producing a standard curve of absorbance versus concentration. By means of measurements at three independent wavelengths, the content of the met, deoxy, and oxy forms of hemoglobin will be determined in a sample of unknown redox composition. In addition, the influence of two different reducing agents on the redox state of hemoglobin will also be determined.

Background and Theory

Optical spectroscopy:
Nelson & Cox. "Lehninger Principles of Biochemistry" p. 80-82—Trp and Tyr spectra
Nelson & Cox. "Lehninger Principles of Biochemistry" pp. 136-139—X-ray and NMR spectroscopy

Hemoglobin:
Nelson & Cox. "Lehninger Principles of Biochemistry" p. 132-135—globin structure and function
Nelson & Cox. "Lehninger Principles of Biochemistry" pp. 157-174—hemoglobin

Redox potentials:
Nelson & Cox. "Principles of Biochemistry" pp. 507-517—oxidation-reduction reactions

References

Lectures

Introduction

Figure 1. Redox states of the heme prosthetic group in hemoglobin.

Hemoglobin is a protein that transports dioxygen (O₂) to cells in vertebrates and in some invertebrate animals. In higher animals, the protein is an α2β2 tetramer. Each of the two types of subunits is a globin polypeptide chain containing a heme prosthetic group. The heme prosthetic group is a protoporphyrin IX molecule complexed with an iron atom (Figure 1). The iron is bound to the porphyrin ring through nitrogens provided by four methylene-linked pyrrole groups. In addition, the heme iron has an axial ligand provided by a histidyl residue from the polypeptide chain. The resting physiological redox state of iron in hemoglobin is ferrous.

Dioxygen reversibly binds to a second axial ligation site on the ferrous iron of the heme moiety. When fully saturated, the tetramer can bind four molecules of O₂ in a reversible, cooperative fashion. In oxy-hemoglobin (the form of hemoglobin carrying O₂), the ferrous iron is oxidized to the ferric state and O₂ is reduced to the superoxide anion (O₂). Upon reversible release of O₂, an electron is returned to the iron to regenerate the functional deoxy-hemoglobin. Occasionally, the ferrous iron of the heme becomes oxidized to the ferric state (met-hemoglobin). Met-hemoglobin does not bind O₂, and is thus in a physiologically inactive state. However, red blood cells have a met-hemoglobin reductase enzyme that can enzymatically reduce met-hemoglobin to the functional ferrous form using NAD(P)H as the reductant. The interconversions of these redox states of hemoglobin are shown in Figure 1.

Hemoglobin is one of the best characterized of all proteins, and UV-visible absorption spectroscopy has been extensively used to study its structure and function. The intense absorption bands of hemoglobin above 320 nm arise from π-π* electronic transitions of the porphyrin molecule (Soret and visible bands). Both the molar absorptivities (ε) and the wavelength maxima (λmax) for these electronic transitions are influenced by the redox state and ligation state of the central iron. As indicated in Table I, met-, deoxy-, and oxy-hemoglobin have different absorption maxima. Consequently, the concentration of each redox state of heme present in a sample of hemoglobin can be determined by measurement of the absorbance at the three wavelengths listed in Table I.

Table I. Molar absorptivity (ε, M-1 cm-1), per heme, of met-, deoxy- and oxy-hemoglobin.

Data Analysis

For mixtures containing many optically active substances, the Lambert-Beer law can be written as:

A = b (1)

In eq 1, A is the measured absorbance at a given wavelength, b is the pathlength of the optical cuvette, ε_i is the molar absorptivity of the i^th substance at the given wavelength, and C_i is the molar concentration of the ith substance. Typically, b is 1 cm, and will not be included in following derivations. For hemoglobin, the total absorbance at each wavelength is the sum of absorbances contributed by each redox state. For this case, eq 1 can be expanded to provide the following three equations:

A₁ = ε₁₁ C₁ + ε₁₂ C₂ + ε₁₃ C₃
A₂ = ε₂₁ C₁ + ε₂₂ C₂ + ε₂₃ C₃ (2)
A₃ = ε₃₁ C₁ + ε₃₂ C₂ + ε₃₃ C₃

If you use the εvalues from Table I, A₁ represents the absorbance at 415 nm, which contains contributions from the met-, deoxy- and oxy-heme redox states. Likewise, A₂ represents the absorbance at 420 nm, and so on. The system of simultaneous equations shown in (2) could be laboriously solved by substitution, i.e., solve equation A₁ for C₁, substitute the result for C1 into the equation for A₂, solve A₂ for C₂, substitute the results for C₁ and C2 into the expression for A₃, and then solve for C₃. The value of C₃ can then be used in the other equations to find values for C₂ and C₁, and so on, until the desired results are obtained. Perhaps you may wish to verify that this type of effort can easily lead to math errors!
Equation 2 can also be represented in a matrix form, as shown below. In this multicomponent formulation of the Lambert-Beer law, the matrix [ A ] contains the absorbance values recorded at three different wavelengths, the matrix [ ε ] contains the molar absorptivities of met-, deoxy-, and oxy-hemoglobin at these three wavelengths (see Table I), and the matrix [ C ] contains the concentrations of met-, deoxy-, and oxy-forms of heme (note that the vertical bars do not indicate absolute value, but are mathematical symbols that indicate the material contained within them is a matrix).

Computer-derived solutions of matrix expressions such as those shown above are a routine laboratory practice. Many hand-held calculators and commercial programs, such as spreadsheets, provide the ability to determine the matrix solutions by a variety of different computational methods. The use of these programs is highly encouraged for data analysis in Biochemistry 651, and a representative Excel spreadsheet that does this calculation is available on the class website in the SPY experiment directory.

A warning to the wise, however, even though your computer algorithm may be coded correctly and provide a self-consistent numerical solution, the noncritical use of computer calculation methods can lead to incorrect interpretation or incomplete understanding of the biochemical and physical phenomena being studied. For this reason, you should always try to corroborate the appropriateness of your experimental results and data analyses with additional evidence supporting or refuting your hypotheses. One way to provide corroborating evidence would be to evaluate your results in the context of previously determined results, including the known biological function. For this lab experiment, you should consider the biochemical properties of hemoglobin as an O2-transport protein, and the limitations of instrumental measurements in making these evaluations.

Procedure

You should work alone on the preparation of the report. Use the DATA SHEET provided in the lab manual to record your results. The Teaching Assistant or Instructor should initial your DATA SHEET when it is completed, before you leave the laboratory. If you use computer software to perform your calculations, indicate what type of computer and software package you have used. In addition, provide a printout of your work that shows the computational approach.

Before beginning the lab:
Review the operating procedures for the spectrophotometers and printers. Be certain that you know how to:
Turn on the spectrophotometer and printer; position the reference and sample cuvettes;
Set the reading mode, scanning speed, and absorbance and wavelength limits.
1. Operation of the spectrophotometer
(a) Turn on the main power to the spectrophotometer and the printer. Allow the spectrophotometer to run through its calibration procedure.
2. Determination of hemoglobin absorption maxima.
(a) Obtain an 8 mL solution of hemoglobin dissolved in 40 mM phosphate buffer (pH 7.5), 10 mL of the same buffer without hemoglobin, a pair of quartz cuvettes, and a pair of plastic cuvettes. Record the hemoglobin protein concentration on the DATA SHEET.
(b) Take a background spectrum (i. e., “zero” the instrument) with buffer in both the reference and the sample cuvettes (see “Single Scan,” step 4F, in the spectrophotometer operating procedures).
(c) Remove the buffer cuvette from the sample compartment and replace it with the solution of hemoglobin. Set the absorbance range on the spectrophotometer from 0.0 to 2.0; set the scan rate to FAST. Scan and PRINT the absorbance spectrum of hemoglobin from 670 to 230 nm.
(d) Read the absorbance values at 415, 420, and 430 nm (see “Fixed Wavelength Measurements,” step 2, in the operating procedures), and record these values in the original sample column of Table V of the DATA SHEET.
(e) Rinse the quartz cuvettes and place them aside in the cuvette holder.
3. Molar absorptivity of hemoglobin in the Soret region.
(a) Place buffer in both plastic cuvettes, and zero the spectrophotometer at 415 nm by pressing the auto-zero button.
(b) Assemble dilutions of the hemoglobin solution according to Table II (see below).

Table II. Hemoglobin standard dilutions.

(c) Measure the absorbance of each sample at 415 nm. Enter the data into TABLE IV of the data sheet.
4. Changes in the hemoglobin spectrum in the presence of ascorbate
(a) Obtain a 1 mL solution of hemoglobin plus ascorbate from your T.A.
(b) Scan and PRINT the spectrum from 670 to 320 nm, and record the absorbance values at 415, 420, and 430 nm in the appropriate columns of TABLE V of the DATA SHEET (as done in Step 2.(d).
(c ) Zoo, in on the spectrum. Reset the absorbance limits from 0.0 to 0.5. Scan and PRINT the spectrum from 670 to 500 nm.
5. Changes in the hemoglobin spectrum in the presence of sodium dithionite
(a) Obtain a 1 mL solution of hemoglobin plus sodium dithionite (sodium hydrosulfite, Na2S2O4) from your T.A. Do not swirl or shake the sodium dithionitereduced solution.
(b) Reset the absorbance limits from 0.0 to 2.0. Scan and PRINT the spectrum from 670 to 320 nm, and record the absorbance values at 415, 420, and 430 nm in the appropriate columns of TABLE V of the DATA SHEET.
6. Finish up.
(a) Have your TA or instructor initial your DATA SHEET.
(b) The instrumental measurements required for this experiment are now finished.

Data Sheet I

Data sheet  [PDF] or [Word doc]

Report

Prepare a cover sheet for your lab report containing your name, section, and title. Include your original DATA SHEET (initialed by a Teaching Assistant before you leave the lab).  In addition, submit one spectrum of each of the three redox states of hemoglobin.  Label these spectra with an identification of the sample, the scan rate, and the wavelength and absorbance ranges.  Label the absorbance maxima of each spectrum with the observed wavelength values.  Label all other absorbance peaks with the absorbance readings and observed wavelength values, as well.  As requested in questions 1 and 2, complete TABLE IV and TABLE VI of the DATA SHEET.  On additional sheets, make the plots for questions 3 and 4 and provide short written answers for questions 5 through 10.

1.    From the known concentration of hemoglobin (concentration of the tetrameric protein) in the standard solution used in PROCEDURE 3.(a), calculate the molar concentration of heme groups in each diluted sample.  Enter these values in TABLE IV of the DATA SHEET. 
2.    Using the information provided in the INTRODUCTION and DATA ANALYSIS sections, and the absorbance values you have entered in Table V, calculate the molar concentrations of met-, deoxy-, and oxy-heme in the standard solution, the ascorbate-treated, and the dithionite-treated samples of hemoglobin.  Enter these values in Table VI of the DATA SHEET. 
3.    Using the data from TABLE IV of the DATA SHEET, make a plot of the absorbance versus concentration of heme.  Determine the slope of the plot, which is the molar absorptivity, ε.  Be sure to indicates units for ε.  How does your calculated εvalue compare with the published values in TABLE I?  If your determined value differs from the published value by more than 3%, propose a reason or reasons for this difference.  Also, perform a linear least squares analysis of the data (linear regression) and report the equation of the best fit regression line in the form of “y = mx + b.”  Report the R² value as well.  If the R² value is less than 0.95, give a reason or reasons for why this might occur. 
4.    In the same plot used for question 3, plot as an overlay the results you would predict if the molar absorptivity determination was performed in a cuvette having b = 2 cm.  Clearly indicate which plot corresponds to the bvalues of 1 cm and 2 cm. 
5.    From the spectrum of the dithionite-treated hemoglobin, calculate the ratio of the absorbance at λmax of the Soret band (320-430 nm) to that at λmax of the visible band (550-560 nm).  Can you determine what the ratio of the molar absorptivities of these two absorption bands would be? 
6.    Calculate ε for the dithionite-treated hemoglobin at the λmax of the visible band. 
Table. III.  E˚' values related to hemoglobin function.

Requirements

SPY Matrix Calculator

SPY Matrix Calculator XLS spreadsheet

Lead Instructor(s)

Michael Levine
Email
5524D Biochemistry
890-0275