Microcomputer-Analyzed Initial Rate Kinetics of the Benzene-Enhanced Unfolding of Myoglobin A Biophysical Chemistry Experiment Merlyn D. Schuh Davidson College, Davidson, NC 28036 Kxperimental biophysical chemistry is a prominenr branch ofrhcmistry that generally receives littlrutrenfion in the undereraduate curriculum due partly . - t o insufficient time and costs of experiments. This paper describes an inex~ e n s i v ebio~hvsicalchemistry experiment, designed t o be completed &I &e lab period, chat introduces students t o the subject of globular protein conformation and microcomputer analysis of initial rate data for the unfolding of proteins. T h e experiment has been used by the author in a junior-year physical chemistry laboratory b u t is also appropriate for the biochemistry laboratory. Background Proteins are polymeric molecules consisting of generally more than 100 amino acid residues t h a t are covalently bonded t o each other through peptide bonds (-CO-NH-). T h e primary structure of a protein may be represented as
in which R; is one of about 20 naturally occurring functional groups and jdesignates the number of amino acid residues in a specific protein molecule. In a n aqueous solution a t physiological pH, the polymer backbone of a globular protein folds into one particular three-dimensional conformation called the native state for which the combined Gibbs free energy of the solute plus solvent is minimal. For several decades biochemists have studied the factors t h a t determine protein conformations and the relationship between protein conformation and physiological function.' T h e traditional method has been t o study the unfolding and refolding of protein molecules induced by the presence and removal, respectively, of an organic denaturant such as urea. Can$ reported t h a t small amounts of aromatic hydrocarbons greatly enhance the rate with which myoglobin unfolds in a n aqueous solution of urea. He used integrated rate equations t o determine the reaction order of the unfolding reaction with respect to the concentrations of added benzene compounds. By comparing the measured reaction order t o the number of aromatic amino acid residues t h a t are closest t h a t pi electron interactions to the heme rine, .. he .proposed . bctween twoaromaticaminoacid residuesand the heme ring were critiral to maintaining.the narivestate conformatim of myoglobin. T h e experiment described herein is based upon the work of C'ann.' Howerrr, instead of using inttgratea rate cxpreisions for t h r dutuanalysis, the method oiinitisl rates is used. T h e large number and complexity of intramolecular interactions within a protein molecule make it likely t h a t the reaction mechanism (and hence the reaction order) cbanae during the unfolding of a protein. T h e advantageof theinitial rates method is to allow determination of the true reaction order by extrapolation t o time zero. ~
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740
Journal of Chemical Education
Materials Myoglobin. Myoglobin was Type I1 Sperm Whale from Sigma Chemeial. Stock solutions of myoglobin with concentrations of 0.35 X 10-&M,0.85X 10-'Mand 1.2 X 10-'Minanaeetate buffer (pH = 5.8. I = 0.05) were oreoared. . . Benrene.Anaqueous,accrotr-boffered snlutionof7.U M urea was weu as rhe iolvent for srock wlur~oncnmtnininc henzcne ronccntrationsofO005 M,0.10 M,0.015 M.0.020 M.andU.025 M. Equipment A Cary 14 UV-visihle spectrophotometer was used to record ahsorbance.However, any suitable visible spectrometer (such as Spectronic 20) is suitable. Method In order to determine the order of reaction with respect to myoglobin concentration, in separate experiments 0.3 mL of each of the three mvoelobin solutions were lavered on top of 2.7 mL of 0.015 M benzendsdution in an absorptionamette. In order to determine the order of reaction with respect to benzene concentration, in separate experiments 0.3 mL of 0.85 X lo-' M myoglobin solution was carefully layered on top of 2.7 mL of the five benzene solutions in an absorption cuvette. Immediately after each cuvette was filled with a benzene solution and myoglobin solution, it was capped, inverted three times to ensure mixing, and quickly placed into the sample compartment of the Cary 14, and absorbance was monitored at 409 nm (the peak of the major Soret absorbance) as a function of time until the absorbance began to level off. The decrease in Soret absorbance is believed to arise from unfolding of the native state, which exposes the heme to the solvent. The concentration of myoblogin, [Mb],was expressed according to eq 1, in which [Mbloisthe concentration of the native-state myoglobin at time zero; and A,, Aa, and A are the Soret absorbance at infinite time, time zero, and time t, respectively. [Mb] = A-AIMbIo Ao- A,
(1)
In accordance with the method of Hall.. Quickenden. and Wattsl. " plots of [Mh] vs. time were fitted hy using a standard polynomiai regression computer program. ~~~~~~~
~
[Mb] = a
+ bt + ct2 + . . .
(2)
a, b, c, etc., are obtained from the best polynomial fit of the data. Differentiation of eq 2 and extrapolation t o t = 0 gives
d'Mbl =
b = -initial reaction rate
(3)
The general rate equation for benzene-enhanced unfolding of myoglobin is
-
= b = h[MbIm[B]" (4) dt in which h is the rate constant for reaction, [B] is the concentration ofhenzene,and rn and nare theorders ofreaction with respectto the
' Chothia, C. Ann. Rev. Biochem. 1984, 53.537.
Cann, J . R . Biochem. 1967, 6, 3435. Hall, K. J.; Quickenden, T. L.; Wans, D.W. J. Chem. Educ. 1976,
53, 493.
Klnetlcs Parameters
benzene absent' benzene Dresent
-
1 0.95
i 0.2
2.1 1.3
0.99 i 0.2
+ 0.4 X
+ 0.2 X
1-'
M-' 5-'
'In theabwnceaf myoglobln a plot of In [Mb] vs. I was linear (firstader) wim s slope of 2.1 i 0.4 X s-'.
l!L:~ -In rate
Flgns 1 Myoglooln mncennatlon vg time (a) b.ffereo solvenl conlil ns 6 3M dea. ib) butterea solvent mntamr 6 3 M urea and 0 0135 M oenzene
16.6
4.0
3.6
3.2
-In [benzene]
- I n rate 17.2
Figure 3. -In rate ~ 6 -In . [benzene].The buffered solutions contain 8.5 X M myoglobin. The slope showsthat the reaction Is first order with respect to [benzene].
17.6 12.6
12.2
11.8
-In [myoglobin]
F l ~ ~ 2.a e-k rate vs. -In lmvmlobinl. . . - . The bufferedsolutions comain 8.3 M wea and 0 0135 M oenzene The slope shows lnat Me react on is flrstorder ~ 0 t le~psct h to [myoglobm]
concentrations of myrglohm and henzene, respectively. Taking the natural logarithm of buth sides of eq I gives m and n are then determined from the slopes of plots of In b vs. In [Mb], with [B] held constant, and in b vs. [B], with [Mb] held constant, respectively. k is determined from the intercepts for these plots. Results
Figure 1shows plots of [Mb] vs. time in the presence of 6.3
M urea (top curve) and in the presence of both 6.3 M urea and 0.0135 M benzene (bottom curve). Figures 2 and 3 show plots of In (reaction rate) vs. In [Mb] and In [B],respectively. The table tabulates the kinetics parameters obtained in typical student experiments. Discussion
The value of 1.0 for m in the table is the same as that obtained by Cannz and is consistent with the view that the decrease i n - ~ o r eabsorbance t corresponds to the unimolecular unfolding of myoglohin. Moreover, a larger value for m would suggest that the interaction between myoglobin molecules was being monitored
Cann reported a value of n = 2 instead of 1as in the table. The difference in results is a . ~.o a r e n t l vdue to the fact that Cann's use of integrated rate expressions produces a reaction order that is measured a t times after t = 0 where the reaction order may actually be changing with time. This difference makes clear the advantage of obtaining the reaction order by extrapolation to t = 0. Consideration of the X-rav crvstalloeranhic structure of myoglobin4 shows two aromkic residues eiose to the heme with roughly parallel orientations of the aromatic and heme rings. The value of n = 1 suggests that the displacement of onlv one of the two aromatic rines from interaction with the heme is the cause for the benzene enhanced unfolding of myoglobin. From the literature4students may locate the two aromatic amino acid residues close to the heme and speculate as to which is more likely to be the one that is critical to the maintenance of the myoglohin conformation and is displaced by benzene. Students benefit educationally from the experiment described here because they: 1. apply the method of initial reaction rates in an experiment where
it is needed, 2. do a microcomputer-assistedanalysis of data, 3. are introduced to the physical biochemistry of proteins, 4. are able to compare their results to work reported in the litera-
ture, and 5. are introduced to the use of a published X-ray crystallographic
structure of myoglobin.
'
Dickerson. R. E.; Geis. I. The Structure and Action of Proteins; Harper and Row: New York. 1969; p 48.
Volume 65 Number 8
August 1988
741
