In the Laboratory

380 Journal of Chemical Education • Vol. 77 No. 3 March 2000 • JChemEd.chem.wisc.edu

Thermal Denaturation of Proteins Studied by UV WSpectroscopyNatas̆a Poklar* and Gorazd VesnaverFaculty of Chemistry and Chemical Technology, University of Ljubljana, As̆ kerc̆eva 5, 1000 Ljubljana, Slovenia;*natasa.poklar@uni-lj.si

It is well known that owing to a direct and simple relation-ship between the concentration of the absorbing species in asolution and its absorbance (Beer–Lambert law), UV–visspectroscopy is mainly used for concentration measurements.In some cases, however, absorption also depends in a sig-nificant way on the molecules’ environment. The change inspectrum with environment can then be interpreted in termsof biological events, such as unfolding of proteins or nucleicacids and binding of ligands to proteins or nucleic acids. Theequilibrium thermal unfolding of α-chymotrypsinogen A atpH 3.0 discussed as a two-state process provides an exampleof how to use UV spectroscopy to follow the temperature-induced unfolding of proteins and how to determine theapparent standard (van’t Hoff ) enthalpy, ∆H°(Td), and theapparent standard entropy of denaturation, ∆S°(Td), at thetemperature of the half-transition, Td.

This experiment is appropriate for an undergraduatephysical chemistry course in which the thermodynamics ofphase transition is studied in detail and for biophysical chem-istry and biochemistry courses where the relation of thermo-dynamics to biological function and activity is emphasized.

Experimental Measurements

The experiment described below does not require anyspecial equipment (only UV–vis spectrophotometer with cellholder) and can be performed in the regular 4-hour laboratoryperiod. Checking the reversibility of the process requires an-other two hours.

α-Chymotrypsinogen A type II from bovine pancreas waspurchased from Sigma (C-4879) as a six-times crystallizedand lyophilized powder and was used without further purifi-cation. One to 2 mg of the protein (weighed on a balance)should be dissolved in 1 mL of 10 mM glycine–10 mM NaClbuffer (pH 3.0). This stock solution of the protein shouldbe diluted with buffer to obtain a final concentration of about0.5 mg mL�1. The final concentration of α-chymotrypsinogenA is determined by applying the Beer–Lambert law at 280 nmand 293 K

Aλ = ελ�c (1)

in which Aλ is the measured absorbance, � is the path lengthin cm, c is the concentration in g mL�1 and ε is the specificabsorptivity, whose value at 280 nm and 293 K is 2000 cm�1

g�1 mL (1).The absorbance of α-chymotrypsinogen A as a function

of temperature was measured at 293 nm. This wavelengthwas chosen because the measured changes of absorbance at293 nm are large and reflect mainly the temperature-inducedexposure of the α-chymotrypsinogen A tryptophan residuesfrom the hydrophobic core of the protein to the aqueousenvironment (2). Sample and reference quartz cuvettes (1-cm

path length) were filled with protein solution and buffer,respectively. Each cuvette has to be sealed with a Teflon stopperto prevent evaporation during the experiment. If a tempera-ture scanning UV spectrophotometer is available, the absor-bance should be recorded as a function of temperature overthe range from 20 to 85 °C at a heating rate of 1 °C min�1.The reversibility of the process should be checked by cool-ing the sample to 20 °C and reheating it to 85 °C (for de-tails see the Lab DocumentationW).

Discussion of Results

By measuring the α-chymotrypsinogen A absorbance at293 nm and pH 3.0 as a function of temperature, a typicalsigmoidal UV melting curve is obtained (Fig. 1). Studentswill now learn how to determine the model-dependent ther-modynamic quantities for a reversible process by analyzingthe α-chymotrypsinogen A melting curve in terms of the two-state approximation (3).

Assuming that temperature-induced α-chymotrypsinogenA denaturation at pH 3.0 is a reversible two-state transition

Figure 1. Absorbance of α-chymotrypsinogen A (in 10 mM glycine–10 mM NaCl buffer, pH = 3.0) at 293 nm as a function of tempera-ture. Concentration of the α-chymotrypsinogen A was 0.5 mg mL�1.The heating rate in experiment was 1 K min�1.

0.40

0.44

0.48

0.52

0 20 40 60 80 100

A29

3

T / °C

∆T = (6.3 ± 0.5)°C

= 1)

AN

= 0)

Td = (55.0 ± 0.5)°C

( = 0.5)

∆Ho (Td ) = (570 ± 30) kJ mol-1

( ∂fD

fD

/ ∂T )Td

= 1/ ∆T

(fD

(fD

AD

In the Laboratory

JChemEd.chem.wisc.edu • Vol. 77 No. 3 March 2000 • Journal of Chemical Education 381

between the native, N, and the denatured, D, state,

K(T )

N ←→ D (2)

the equilibrium constant K(T ) can be expressed as

K T =aDaN

(3)

where aN and aD are the protein activities in its native anddenatured states, respectively.

Such model transition can be described in terms of experi-mental variables (observables such as absorbance, viscosity,fluorescence, heat capacity, etc.) in a very simple manner. If ata given temperature y(T ) is the value of the observable usedfor following the thermal transition of the protein and yN(T )and yD(T ) are the corresponding observable values of theprotein in its initial (native) and final (denatured) state, thenthe transition can always be described as (3, 4):

y (T ) = f N(T )y N(T ) + f D(T ) yD(T ) = y N(T ) + f D(T )[ yD(T ) – y N(T )]

(4)

where fN(T ) + fD(T ) = 1, and fD(T ) and f N(T ) representthe fraction of molecules in the native and denatured state,respectively. Since students follow the absorbance, A, at achosen wavelength as a function of temperature, then y (T ) =A(T ) and the fraction of the thermally denatured protein,fD(T ), at a given temperature takes the form

fD T =y T – yN T

yD T – yN T=

A T – A N T

A D T – A N T (5)

in which A(T ) refers to the measured absorbance at tempera-ture T, and AN(T ) and AD(T ) refer to the correspondingabsorbance values of the native and denatured state at thesame temperature. The values of AN(T ) and AD(T ) can beobtained at any point in the transition region by extrapolationfrom the linear portions of the measured UV melting curve(Fig. 1). Since at any stage of the transition the concentra-tions of native state, [N], and denatured state, [D], can beexpressed in terms of the total protein concentration, c0, as[N] = c0(1 – fD) and [D] = c0 fD, the resulting apparent equi-librium constant, K°app(T ) takes the form

Kappo T =

D

N=

fD T

1 – fD T=

AN T – A T

A T – AD T(6)

(NOTE: This is not a true equilibrium constant, because theconcentrations of the molecules in the native and denaturedstate are used instead of their activities. When dealing withprotein solutions we are forced to use apparent thermody-namic quantities because the activity coefficients and thusactivities of the solute species are not known.)

The apparent standard molar Gibbs free energy of de-naturation, ∆G °(T ), can be obtained from the apparent equi-librium constant, K °app(T ) as

∆G°(T ) = �RT ln K °app(T ) (7)

where R is the gas constant. The corresponding apparent stan-dard enthalpy of denaturation, ∆H°(T ), follows directly from

the Gibbs–Helmholtz relationship:

∂∆G° T

T∂T P

= �∆H° T

T 2(8)

which can be further transformed, using eqs 7 and 8, intothe van’t Hoff relation:

∆H° T = RT 2

d ln Kappo

dT(9)

At the temperature of half-transition, Td, defined as thetemperature at which half the protein molecules are in thenative state and half are in the denatured state ( fD = fN =0.5), eqs 6 and 7 result in K °app(Td) = 1 and ∆G°(Td) = 0.Furthermore, by applying eqs 6 and 9 it is easy to show thatat T = Td the van’t Hoff enthalpy of denaturation (eq 9) takesthe form

∆H° Td = 4RTd2 dfD

dT T d

(10)

As shown in Figure 1, the derivative (df D/dT)Td can beapproximated from the UV melting curve as (dfD/dT)Td

=[(fD = 1) – (fD = 0)]/∆T = 1/∆T where ∆T is the temperatureinterval in which the two-state approximation of the thermalunfolding is completed (Fig. 1). Thus, the van’t Hoff relation(eq 10) can be further simplified into

∆H° Td = 4RTd2 1∆T

(11)

The ∆H°(Td) for α-chymotrypsinogen A at pH 3.0 obtainedfrom eq 11 using the slope of the measured melting curvepresented in Figure 1 has a value of (570 ± 30) kJ mol�1 at328 K, which agrees well with the literature data (5, 6 ).

Finally, since ∆G°(Td) = 0, one can express the apparentstandard entropy of denaturation at T = Td , simply as

∆S° Td =∆H° Td

Td(12)

The entropy value, ∆S °(Td), for α-chymotrypsinogen Aat pH 3.0 calculated from eq 12 has a value of (1.7 ± 0.1)kJ mol�1 K�1.

The students will find that both ∆H°(Td) and ∆S °(Td)are positive, meaning that the process of thermal denaturationof α-chymotrypsinogen A at pH 3.0 and 328 K is endothermic(∆H > 0 ) and disordering (∆S > 0). They will get acquaintedwith the general picture of the denaturation process accordingto which the observed endothermic ∆H °(Td) values resultfrom endothermic contributions due to the intramolecularhydrogen bonding, van der Waals interactions, and hydrationeffects, while the positive ∆S °(Td) values are due to theprevalence of the conformational contribution (∆Sconf > 0)over the contribution due to the hydrophobic hydration ofthe unfolded protein (∆Shyd < 0) (7, 8).

In conclusion, it should be pointed out that the van’tHoff enthalpies of protein denaturation obtained from UV

In the Laboratory

382 Journal of Chemical Education • Vol. 77 No. 3 March 2000 • JChemEd.chem.wisc.edu

melting curves are model-dependent (3), and to determine thecorresponding model independent values the temperature-induced protein denaturation should be followed by directcalorimetric measurements (9).

WSupplemental Material

Supplemental material for this article is available in thisissue of JCE Online.

Literature Cited

1. Wilcox, P. E.; Cohen, E.; Tan, W. J. Biol. Chem. 1957, 228, 999.

2. Herskovits, T. T.; Laskowski, M. Jr. J. Biol. Chem. 1962, 237,3418.

3. Schellman, J. F. Comp. Rend. Trav. Carlsberg Lab., Ser. Chim.1955, 29, 230.

4. Lapanje, S. Physicochemical Aspects of Protein Denaturation;Wiley-Interscience: New York, 1978; p 187.

5. Poklar, N.; Vesnaver, G.; Lapanje, S. J. Prot. Chem. 1995, 14,709.

6. Chalikian, T. V.; Völker, J.; Anafi, D.; Breslauer, K. J. J. Mol.Biol. 1997, 274, 237.

7. Makhatadze, G. I.; Privalov, P. L. J. Mol. Biol. 1993, 232, 639.8. Privalov, P. L.; Makhatadze, G. I. J. Mol. Biol. 1993, 232, 660.9. Chowdhry, B.; Leharne, S. J. Chem. Educ. 1997, 74, 236.