Protein Denaturation and Tertiary Structure Janice S. Barton Washburn University, Topeka, KS 66621

Protein conformation is conveniently divided into four levels of structure-primary, secondary, tertiary, and qua- ternary. The sequence of amino acids constitutes primary structure, helix and sheet configurations compose secondary structure, and folding of the linear polypeptide chain (sec- ondary structure) upon itself to create the three-dimension- al structure of a single chain constitutes tertiary structure. A protein with mul t i~ le chains Dossesses auaternarv structure. wi th the exceptioh of the str;cture le;els are dis- rupted hy denaturation with a variety of chemical reagents. Alterations of secondary structure are readily monitored by circular dichroism and optical rotatory dispersion. NMR spectroscopy, fluorescence spectroscopy, and viscosity are among the many techniques that facilitate observation of changes in tertiary and quaternary structure.

Although this concept of protein structure is dutifully learned bv manv students. its meanine is m i t e often ab- .. . stract d u i t o thejack of opportunity for ohserving thephysi- cal entity. 'l'o hridre this EaD. this inexpensive denaturation experiment is designed 6 demonstrate the presence of ter- tiarv structure through observation of changes in this struc- . - ture with viscosity measurements. Bovine serum albumin, being relatively inexpensive, is used to demonstrate tertiary structure by comparing the appropriate viscosity parame- ters for three states of the protein-the native, the dena- tured, and the reduced and denatured protein.

Alterations in tertiary stnrcture are monitored by viscosi- ty, which is sensitive to the overall conformation1, and re- duced viscosities are compared for the three states of this protein. The dynamic viscosity, nlp, is related to the flow time in a capillary viscometer, and t = k d p where t is the flow time, n is the viscosity, p is the density, and k is the viscometer constant. By using the same viscometer for solu- tion and solvent, the relative viscosity is given by (tlto = n.poln,yp) with the naught denoting solvent. With dilute solutions, the density of solution aproximates that of the solventz, the relative viscosity becomes tho, and the specific viscosity (n,) is (tlto - 1). I t is the reduced viscosity (n&) that is so often related to the intrinsic viscositv. expressed in - . . terms of molecular volume and geometry. The reduced vis- cositv is therefore used t o assess the effect of denaturation and the reduction of disulfide bonds on the tertiary struc- ture of albumin.

Experimental A 10 to 40.0 mgfml solution of bovine serum albumin in 0.1 M

sodium phosphate, pH 7.2, is supplied to the students. The albumin of 98-99% purity can be purchased from Sigma Chemical Company, catalog #A7906. Ostwald viscometers with flow times of 80 to 100 s for water are employed for viscosity measurements carried out in water baths thermostatted at 25 i 0.2'C. The criterion for aecept- able data is three consecutive flow times that differ by no more than

'Tanford, C.; Kawahara, K.; Lapanje, S. J. Biol. Chem. 1966, 241, 1921.

2Billingham, N. C. "Molar Mass Measurements in Polymer Scien- ce"; Wiley: New York. 1977: p 175.

3Martin, R. B. "Introduction to Biophysical Chemistry"; McGraw- Hill: New York. 1964; pp 172. 239.

'Haschemeyer, R. H.; Haschemeyer, A. E. "Proteins"; Wiley: New York. 1973; p 177.

5Tanford, C.; Buzzell, J. G. J. Phys. Chem. 1956, 60, 225.

Reduced Vlrcosltles*

Conformational State n,/c i s.d. (mlfg) N

native 4.8 i 1.1 18 denatured, pH 2 9.7 i 2.2 i9 reduced. denatured 13.1 f 4.5 18

O S s lor each uirhs three states: nativealbuminat pH 7.2, denatured alhmin at pH 2, and redwed and denatured alb~~min at pH 2 and ICo beta-mercnptoethonol. I\ separate 5-ml sample is employed fnr the viscosity measurement on each of the three states of albumin because of the tendency of protein to stick to the glass, to bubble, and to denature with repeated movement through the capillary. Analog or digital stopwatches with sensitivities of 0.1 and 0.01 s, reanectivelv. can he used to measure flow times. ...r. ~~~ ~

An nlhumin solution oi pH 2 with aconcentration between 10 and 40 mg ml rs arhieeed by adding 6 N HC1 dropwivr with stirring. For a 10-ml sAni,,n. only a iw tenthsof a milliliter oiaeid is needed to reach pH 2, andthe ;H value is confirmed using a pH meter with a combination electrode. Beta-mereaptoethanol is added to 1% on a volume basis to reduce disulfide bonds. Beta-mercaptoethanol, a poison with the smell of stench, should he dispensed in a hood, and the portion of the experiment in which it is used should be carried out in a hood or well ventilated room.

Viscometers are cleaned with cleaning solution after being rinsed with water or buffer to remove excess protein. Cleaning is accom- plished between samples and the viscometers are rinsed with dis- tilled water and thoroughly dried. To allow time for students to become familiar with viscometer use, two to three laboratory peri- ods of 3 h duration should be allowed for completion of this experi- ment.

Results and Dlscusslon Twenty-two students reported increases in the reduced

viscosity when albumin was denatured. Nineteen of these students also observed viscosity increases when the disulfide bonds were reduced. These results are consistent with the behavior of a globular protein having disulfide bonds 3.4, as hovine serum albumin is classified ' ,4,5.

Reduced viscosities are reported for albumin concentra- tions of 9.6.20.0.24.0. and 40.0 mglml. These concentrations evinced no statistically significant differences in reduced viscositv when com~arisons were made within each state of albumin. However, comparison of viscosities between states ~roduced significant differences. With carefully controlled conditions, a researcher would expect to observe a concen- tration dependence of reduced viscosities; however, low flow times and lack of tight temperature control (f 0.01") would contribute to sufficient variation in flow times that would tend to blend the distinctions between protein concentra- tions.

Because of this lack of distinction, all of the data for each state of the protein was averaged without regard to concen- tration, except for outlying values. The averages presented in the table, when compared by the t-test, are statistically different from each other a t the 99% confidence level. These data clearly demonstrate the disruption of tertiary structure at pH 2 and the further expansion of the protein when the disdfide restraints are broken.

Volume 63 Number 4 April 1986 367

Denaturation of albumin could also be accomplished in 8 Concluslon M urea or 6 M guanidine hydrochloride, which could be This experiment, which was carried out by two sophomore dialyzed away to measure regeneration of tertiary structure. classes, successfully demonstrates qualitatively and quanti- HCI was selected for this experiment, because the students tatively that changes in tertiary structure accompany pro- found denaturation with i t an easy task. tein denaturation.

368 Journal of Chemical Education