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 quaternary. The sequence of amino acids constitutes primary structure, helix and sheet configurations compose secondary structure, and folding of the linear polypeptide chain (secondary structure) upon itself to create the three-dimensional structure of a single chain constitutes tertiary structure. A protein with m u l t i ~ l chains e Dossesses auaternarv structure. w i t h the exceptioh of the str;cture le;els are disrupted 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 abstract d u i t o thejack of opportunity for ohserving thephysical entity. 'l'o hridre this EaD. this inexpensive denaturation experiment is designed 6demonstrate the presence of tertiarv structure through . observation of changes - in this structure with viscosity measurements. Bovine serum albumin, being relatively inexpensive, is used to demonstrate tertiary structure by comparing the appropriate viscosity parameters for three states of the protein-the native, the denatured, and the reduced and denatured protein. Alterations in tertiary stnrcture are monitored by viscosity, which is sensitive to the overall conformation1, and reduced 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 solution 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. in - . expressed . terms of molecular volume and geometry. The reduced viscositv is therefore used t o assess the effect of denaturation and the reduction of disulfide bonds on the tertiary structure 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 aeceptable data is three consecutiveflow 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 Science"; Wiley: New York. 1977: p 175. 3Martin, R. B. "Introduction to Biophysical Chemistry"; McGrawHill: 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 native denatured, pH 2 reduced. denatured
n,/c i s.d. (mlfg)
N
4.8 i 1.1 9.7 i2.2 13.1 f 4.5
i9
18 18
O S s lor each uirhs three states: nativealbuminat pH 7.2, denatured at pH 2 and alhmin at pH 2, and redwed and denatured alb~~min ICo beta-mercnptoethonol. 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, can he used to measure flow times. reanectivelv. ...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-mlsAni,,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 accomplished between samples and the viscometers are rinsed with distilled water and thoroughly dried. To allow time for students to become familiar with viscometer use, two to three laboratory periods of 3 h duration should be allowed for completion of this experiment. \I
~~~
~
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 concentrations of 9.6.20.0.24.0. and 40.0 mglml. These concentrations evinced no statistically significant differences in reduced viscositv when com~arisonswere made within each state of albumin. However, comparison of viscosities between states ~ r o d u c e dsignificant differences. With carefully controlled conditions, researcher would expect to observe a concentration dependence of reduced viscosities; however, low flow times and lack of tight temperature control (f0.01") would contribute to sufficient variation in flow times that would tend to blend the distinctions between protein concentrations. Because of this lack of distinction, all of the data for each state of the protein was averaged without regard to concentration, 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 p H 2 and the further expansion of the protein when the disdfide restraints are broken.
a
Volume 63
Number 4
April 1986
367
Denaturation of albumin could also be accomplished in 8 M urea or 6 M guanidine hydrochloride, which could be dialyzed away to measure regeneration of tertiary structure. HCI was selected for this experiment, because the students found denaturation with i t an easy task.
368
Journal of Chemical Education
Concluslon This experiment, which was carried out by two sophomore classes, successfully demonstrates qualitatively and quantitatively that changes in tertiary structure accompany protein denaturation.
