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Keywords:

  • Instrumentation;
  • fluorescence;
  • isothermal titration calorimetry;
  • lysozyme

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. DISCUSSION
  5. REFERENCES

Our first-semester biochemistry course is accompanied by a project-based laboratory with a focus on the enzyme lysozyme. The first half of the semester involves purification of the enzyme. In the second half, students learn to work with advanced instrumentation techniques, specifically fluorescence spectroscopy and isothermal titration calorimetry. These methods are linked, as much as possible, to the theme of lysozyme: its structure, stability, or binding of ligand. As a final project, students design an experiment involving one of these methods. Examples are determination of the pKa of the catalytic residue and the binding affinity for an inhibitor under different conditions of pH and temperature. Four weeks of laboratory are sufficient for students to learn how to use the instruments and to develop a short project. At the end of the project, students give an oral presentation on the theory of the method and their results and prepare a paper that undergoes peer review. Hands-on experience with these methods reinforces theoretical concepts taught in the lecture portion of the course.

We have previously described a project-based laboratory to accompany the first semester of biochemistry [1, 2]. The focus of the laboratory is the purification and study of the enzyme lysozyme from hen egg white (HEW).11 Since the time when this laboratory was first designed we have found that students come into the course with more experience in basic biochemical techniques so there is less need for a project on simple enzyme kinetics and protein assay. At the same time, the College has acquired a number of sophisticated instruments that students are unlikely to have encountered in biology courses. We have, therefore, incorporated a 6-week project (4 weeks of laboratory work and 2 weeks for preparation and presentation of results) that introduces the student to more advanced instrumentation, namely fluorimetry and isothermal titration calorimetry (ITC).

The objectives of introducing these project are: 1) to teach methods of protein analysis using advanced instrumentation and 2) to allow students to design their own experiments and then interpret their own data.

Students work in pairs and first spend 1–2 weeks learning to use each of the instruments following a prescribed procedure. They are then asked to build on their knowledge and design their own well researched project. Consultation with the lab instructor reigns in overly ambitious projects and ensures a properly planned experiment. At the end of the 4-week period, each group of students gives an oral presentation on both the theory of the method and their results. This is considered to be a “fun” end-of-term project and reinforces theoretical concepts introduced during the lecture portion of the course.

EXPERIMENTAL PROTOCOL

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. DISCUSSION
  5. REFERENCES

Fluorescence Spectroscopy [3]—

Fluorescence is the emission of radiation that occurs when a molecule in an excited singlet electronic state returns to the ground state by re-emitting light of longer wavelength than that absorbed. The quantum yield describes the probability of fluorescence and is influenced both by properties of the molecule itself and by environmental factors. Quenching, a reduction in the quantum yield, is often used as a measure of the environment in which the fluorophore is found.

Large, flexible molecules are likely to undergo non-radiative transitions, where all absorbed energy is dissipated as heat, so that fluorophores are relatively rare in biological systems. Proteins contain only three intrinsic fluorophores, the aromatic amino acids. Of these, tryptophan is the most widely studied because phenylalanine and tyrosine have lower quantum yields.

As the introductory exercise, students determine the optimal wavelengths of emission and excitation for free tryptophan and follow changes in wavelengths and intensity with changing solvent [4]: Trp is prepared as a 1 μM solution in 0.01 M phosphate buffer, pH 6.0. An emission spectrum is run, scanning from 280 to 500 nm with an excitation wavelength of 280 nm. Using the optimal wavelength from this scan as the emission wavelength, an excitation spectrum is then run, scanning from 250 to 500 nm. The influence of solvent polarity is studied by changing the solvent to methanol with determination of emission and excitation optima and intensity as above.

One independent project that has been pursued by students is to examine the pKa of the catalytic residue of HEW lysozyme, Glu-35. Tryptophan fluorescence is quenched by neighboring protonated acidic groups. Therefore, the pKa of neighboring groups can be determined by measuring tryptophan fluorescence as a function of pH. From the crystal structure of hen egg white lysozyme, it is known that Trp-108 is in van der Waals contact with Glu-35, whose carboxyl group is essential for catalysis. The pKa of Glu-35 in the lysozyme-(NAG)3 complex can be determined by fluorescence. The complex, rather than free enzyme, must be used since the fluorescence intensity of free lysozyme is insensitive to the ionization of Glu-35 [5].

For determination of pKa, lysozyme is made up as a 1.5 × 10−6M solution in 2 mM acetic acid, 2 mM phosphoric acid, 0.1 M KCl. The inhibitor (NAG)3 is added to a final concentration of 1.2 × 10−4M. This sample is adjusted to pH 3 with HCl and then titrated with 0.5 M NaOH. Fluorescence is monitored at constant wavelength, recording the intensity at frequent intervals [5]. The titration curve of the lysozyme-(NAG)3 complex generated (Fig. 1) very clearly demonstrates the unusually high pKa for Glu-35. Careful titration with gradual increments in pH reproducibly (n = 4) showed a pKa between 6.4 and 6.5 for the lysozyme-(NAG)3 complex that agrees with an earlier study [5]. Such results provide an opportunity for discussion of the effect of the hydrophobic environment on ionization and the role of Glu-35 as a proton donor during catalysis.

Students have also examined the stability of lysozyme using denaturants by fluorimetry. A similar experiment has recently been described by Kuritan and Lee [6]. As the concentration of denaturant is raised, protein unfolding is detected as an increase in fluorescence intensity due to the transfer of buried tryptophan residues from the hydrophobic interior to the aqueous exterior.

Isothermal Titration Calorimetry [7]—

Heat is sometimes called the universal detector of biological processes and binding phenomena. ITC is a technique that provides complete thermodynamic analysis to interpret the molecular forces at work in a system. A syringe incrementally titrates ligand into a sample cell containing the protein or other macromolecule. On interaction, heat is released or absorbed, measured by the instrument, and plotted versus time. As the sample macromolecule becomes saturated with ligand, the heat signal diminishes until only background heat from dilution is observed. From a single experiment, the following information about the binding process can be obtained: equilibrium constant, binding stoichiometry, enthalpy of binding, entropy of binding, and free energy change of binding.

In this introductory series of experiments, the goal is to investigate the binding of a ligand, (NAG)3, to lysozyme. N-Acetylglucosamine oligosaccharides less than five subunits long are hydrolyzed very slowly by the enzyme and act as competitive inhibitors. During the 1st week, students learn how to use a Microcal VP-ITC instrument by evaluating the binding of (NAG)3 to lysozyme. Both inhibitor and protein are prepared in 0.1 M phosphate buffer adjusted to the optimal binding pH of 5.0. It is essential that the buffer for both protein and ligand be identical, or the heat of mixing will obscure experimental heats of binding. The inhibitor stock solution is 2.66 mM, and the protein stock solution is 0.14 mM. Approximately 10 ml of lysozyme and 0.5 ml of inhibitor are needed. Both solutions are degassed prior to filling the sample chamber and syringe. Lysozyme is placed in the sample chamber, and ligand is placed in the syringe. Temperature is set at 25 °C. Injections of 10 μl are delivered every 5 min for a total of 25 injections.

After the experiment is complete data analysis is accomplished with Origin software, also supplied by Microcal. A base line is generated, all peaks are integrated, and a curve is fit for a model with one binding site. For the best-fit curve, thermodynamic parameters are calculated and displayed. Five trials by different student groups yielded a binding constant of 1.1 × 105M−1. Sample data are shown in Fig. 2. This average value compares well to binding constants derived by another method, UV difference spectroscopy. Two other studies also conducted at a pH of 5 and temperature of 25 °C yielded values of 1.0 × 105 and 1.3 × 105M−1 [8, 9]. Students who choose to explore ITC further in their independent projects often examine the effects of temperature or pH or compare lysozymes from different species. Lowering the pH, raising the temperature, or use of turkey lysozyme resulted in a drop in binding affinity when compared with HEW lysozyme at a pH of 5 and temperature of 25 °C (Table I).

DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. DISCUSSION
  5. REFERENCES

Our two-step approach ensures that sufficient data are gathered to write a six- to eight-page paper, but it also allows student input and initiative. The introductory weeks enable each student to become familiar with the method and instrument, and this first experiment already yields a wealth of data. The students then choose a parameter to vary to expand their study of the active site of lysozyme. Students must understand the intricacies of the active site of lysozyme to design an interesting project and then to interpret their results. For example, during the examination of ITC results, students can attribute the decrease in binding affinity upon reduction of the pH to 2 to the protonation of Asp-101 and a loss of hydrogen bonding between enzyme and substrate [10]. Asp-101 is also a critical active site residue when the binding affinities of hen and turkey egg white lysozymes are compared. A reduction in binding affinity can again be linked to a loss of hydrogen bonding between turkey lysozyme and substrate since a Gly residue is found in the turkey enzyme in place of the Asp-101 residue of HEW [9, 11]. Additionally, the progressive decline in binding affinity as the temperature is raised above 15 °C can be linked to a conformational transition that occurs between 20 and 30 °C [1214]. Analysis by 13C-NMR has localized a conformational change in the binding site near subsites D and E [13].

The fluorimetry experiment, on the other hand, requires that the student be knowledgeable about the catalytic mechanism and the residues responsible for maintaining a high pKa for Glu-35. The hydrophobicity of Trp-108 and the electrostatic interaction with Asp-52 allows an un-ionized Glu-35 to serve as a proton donor during catalysis [5].

As basic biochemical techniques appear earlier and earlier in students' training and as research opportunities become more accessible, there is a need to introduce state-of-the-art instrumentation into undergraduate biochemistry courses. Although the specific equipment used for the experiments described here may not be available at all institutions, these examples of undergraduate laboratory exercises may suggest other instrumentation projects. As described, the instrumentation project comprises 6 weeks: 1–2 weeks are spent on introductory exercises, 2–3 weeks are spent on a project of the students' design, 1 week is for writing a scientific paper, and 1 week is for presentation to the class. This format allows students more experience in experimental design, library research, peer review and revision, and presentation skills.

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Figure FIGURE 1.. Effect of pH on fluorescence intensity of the lysozyme-(NAG)3complex. A 1.5 × 10−6M HEW lysozyme solution and 1.2 × 10−4M (NAG)3 was prepared in a 0.1 M KCl, 2 mM acetic acid, and 2 mM phosphoric acid buffer. The fluorimeter was set to an excitation wavelength of 280 nm and an emission wavelength of 340 nm. Fluorescence intensity (a.u., absorbance units) and pH measurements were recorded for the lysozyme-(NAG)3 solution (2.2 ml) and after each addition of 2–5 μl of 0.5 M NaOH. In the figure, the first derivative of the curve is shown below the data for fluorescence intensity versus pH, allowing identification of the steepest rise. Thus, the pKa of Glu-35 in the lysozyme-(NAG)3 complex was assigned a value of 6.5.

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thumbnail image

Figure FIGURE 2.. Binding curve for HEW lysozyme titrated with the (NAG)3inhibitor. Isothermal titration calorimetry was carried out at 25 °C with 0.14 mM HEW lysozyme titrated with 25 injections of 10 μl of 2.66 mM (NAG)3. Enzyme and substrate were both prepared in a 0.1 M phosphate buffer, pH 5.0. Data on heat released with each injection were analyzed with Origin software (Microcal).

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Table Table I. Determination of affinity constants for (NAG)3 binding to lysozyme
A. Effects of pHa  
MethodAffinity constant, KpH
 M−1 
ITC (this study)1.1 ± 0.13 × 1055
Kumagai et al. [9]1.3 × 1055
ITC (this study)3.8 × 1043
Banerjee and Rupley [10]3.6 × 1043
B. Effects of temperatureb  
MethodAffinity constant, KTemperature
 M−1°C
ITC (this study)4.0 × 10515
 (3.5–4.5 × 105) 
Banerjee and Rupley [10]4.5 × 10515
ITC (this study)1.1 ± 0.1 × 10525
Kumagai et al. [9]1.3 × 10525
ITC (this study)3.7 × 10445
Banerjee and Rupley [10]3.9 × 10445
  • a

    a All studies were conducted at a temperature of 25 °C using HEW lysozyme.

  • b

    b All studies were conducted at a pH of 5 using HEW lysozyme.

  • c

    c All studies were conducted at a pH of 5.

  • d

    d Study conducted at 25 °C.

  • e

    e Study conducted at 30 °C.

Shown is a comparison of ITC results to those from previous studies that used spectrofluorometric techniques. Standard deviation is included for the value at pH 5, 25 °C where multiple student groups carried out the same study. (Note that this value is repeated in parts A, B, and C for comparison.) In cases where the number of replicates was small, a range is given below the mean. In a few cases of independent projects, only a single student group's result is given.
MethodAffinity constant, KSpecies
 M−1 
ITC (this study)1.1 ± 0.1 × 105Hend
ITC (this study)1.7 × 104Turkeyd
 (1.4–2.0 × 104) 
Banerjee and Rupley [11]3.6 × 104Turkeye
  • 1

    Funding for instrumentation was provided by grants from the National Science Foundation-Instrumentation and Laboratory Improvement program and Howard Hughes Medical Institute; student support was funded by the National Science Foundation Award for Integration of Research and Education.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. DISCUSSION
  5. REFERENCES
  • 1
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  • 3
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  • 4
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  • 5
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  • 6
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  • 7
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  • 10
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  • 11
    S. K. Banerjee, J. A. Rupley (1975) Turkey egg white lysozyme. Free energy, enthalpy, and steady state kinetics of reaction with N-acetylglucosamine oligosaccharides, J. Biol. Chem. 250, 82678274.
  • 12
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  • 13
    P. J. Cozzone, S. J. Opella, O. Jardetzky, J. Berthou, P. Jolles (1975) Detection of new temperature-dependent conformational transition in lysozyme by carbon-13 nuclear magnetic resonance spectroscopy, Proc. Natl. Acad. Sci. U.S.A. 72, 20952098.
  • 14
    J. Saint-Blanchard, A. Clochard, P. Cozzone, J. Berthou, P. Jolles (1977) The temperature-dependent structural transition of lysozyme. A study of the Arrhenius plots, Biochim. Biophys. Acta 491, 354356.