I 30 3
0.00 20
tb
(rnl",
Figure 2. Type ii Kinatic Experiment. AA, and AA . data far each cuvene are used lo establish the scale indicated in Uis figure. Data listed as a, b, c, and don the graph represent time paints (50 fii aiiquds) taken after iniliation of the incubatbn reenion mixtlre. These inwbstion times in *is pariicvlar expwiment were a = 0.21 min, b = 3.67 mi", c = 5.20 mi". andd = 11.50 min. Conditions and AAmhcontrois as well as the incubation reaction mixture are for AA, given in the text.
Type II Klnetics
Figwe 3. Guggenheim Plot for kinetic reaction " d of Figure 2.
Sample and reference cuvettes are again washed and fded with proflavin solution, equilibrated a t 25"C, and the baseline absorbance of each cuvette is recorded. Then, an incubation mixture of a-Ct and sultone is formed (see below) and a timer is activated to monitor incubation time. At timed intervals, a 60 p1 aliquot of the incuhation reaction mixture is taken and quickly added with stirring to one of the sample cuvettes and the instantaneous absorbance is recorded. With the aid of - - the ~--. &loo% free enzyme scale established for the cuvette, the instantaneous absorbance change of the incubation reaction mixture aliquot gives the percentage of non-EA enzyme detected by the assay. Since many spectrophotometers have room for four sample cuvettes, students are able to eet four timed measurements of the instantaneous absorhanc; from an incuhation reaction mixture. For the fourth and final cuvette. the instantaneous absorbance a t 465 nm is recorded but instksd of stopping the recording, the absorbance is followed for 20 min. This tracing follows first order kinetics (see Fig. 2) and the rate constant is determined from a Guggenheim plot (10,ll) (see Fig. 3). The compositions of the incubation control mixtures, AA,, and AA,i,, as well as the incubation reaction mixture are as follows: (1) AA,, incubation control mixture consists of 250 pl of 0.1 M citrate buffer, pH 7.0 mixed with 50 fi1 of acetonitrile. This is followed by addition and mixing of 50 pl of 60 mg/ml a-Ct dissolved in pH 3.0 HCl. (2) M,,,incubation control mixture is composed of 250 p1 of 0.1 M citrate buffer, pH 7.0, mixed with 50 p1 of 28 m M sultone I in acetonitrile. This is followed by addition and mixing of 50 p1 of pH 3.0 HCl. (3) The incubation reaction mixture starts with 250 pl of 0.1 M citrate buffer, pH 7.0, mixed with 50 pl of 28 m M sultone I in acetonitrile, 25'C. This is quickly stirred with 50 PI of 60 mglml a-Ct a t 25% concomitant with starting a timer to monitor incubation time. All three incuhation mixtures have a total volume of 350 p1 each.
The objective here is to quantitate the amount of intermediate (EAJ which accumulates on incubating a-Ct with sultone I. On mixing an aliquot of such an incuhation mixture into a solution containing excess proflavin, all non-EAforms of the enzyme ultimately bind proflavin a t their active sites and increase the 465 nm absorbance (9). EA, on the other hand, does not bind proflavin and will not register a 465 nm absorbance increase. Thus, in order to quantitate the percentage of total enzyme in the form of sulfonylenzyme (EA), it is necessarv to establish the ranee of absorbance chances expected if: (i)no EA is formed in &e incubation mixture; I:e,. all of the enzyme binds to proflavin upon assay (AA,,.) and ('2) all of the enzyme gets tied up as sulfonylenzyme and no enzyme is detected by the proflavin assay ( M & . This means that we must sample from three incuhation mixtures. One is the AA,;, control incubation mixture in which no proflavin-enzyme'binding is possible. The second is a M,., control incubation mixture in which all of the enzyme isdZcted hy mmplexation with proflavin. And the final one is the incubntion mixtureof rr-Ct andsultone which eives an absorbance change proportional to the amount of ~OG-EA enzyme present. The compositions of these three incubation mixtures are given at the end of this section. The protocul our students have successfully used for four years requires careful maintenance of temperature and individual calibrations of the cuvettes used. The reference and four sample cuvettes are filled with 3.0 ml of 120 pM proflavin solution (0.1 citrate buffer, pH 7.0). These are stoppered and equilibrated for 10 min in the spectrophomneter at 25.0 f O.l°C. The absorbance baselines of the four sample cuvettes nm and are set to ahout 4090of the 0.1 ahsorhance scale at 465 ~-~ -~ ---.--Results and Dlscusslon the absorbance of each cuvette is registered on the chart. AA,, for each sample cuvette is recorded after adding 50 p1 The work described here is performed over a two-week of the Urn,control mixture (also controlled a t 25°C) to each period ( 4 hours total) in the laboratory accompanying our cuvette. The samole and reference cuvettes are then waqhed. two-semester biochemistry course. The lab consists of a lecrefilled with proflavin solution as above, incubated a t 2 5 " ~ ture, difference spectrum, and t w e I kinetics oerformed one for 10 min in the soectroohotometer and a baseline is aeain week, and type I1 kinetics haddled the second week. The recorded for each cuvette: M,, is recorded for each cnvette methods used are made possible bv the kinetic characteristics after mixine in 50 ul of the M-i. control mixture to each of of a system which define a rather "nique interaction between the cuvettei. At this point the &orbance scale for detecting a-Ct and sultone I. 070 to 100Dofree enzvme (non-EA) is established for each cuWe have previously shown that eqn. (1) faithfully describes . vette (see Fig. 2). Because of the high absorbance of proflavin the a-Ct mediated hydrolvsis of sultone I and that the interin samole and reference cuvettes. the difference absorbances mediate (EA) which acc&ulates is a covalent sulfonylenzyme are hiihly sensitive measuremenk For best results in repro(66). The ahility of EA to revert to sulwne is quite sienificant ducibilitv of AA,. and AA,;. values. each cuvette must be with k-z several fold greater than k s (60). ~bnsequintly,in carefully-washedafter each n&uremeh. Particular attention the steady state, ES is in virtual equilibrium with EA. must he paid to accuracv in pipettine. Even with care, the The fvst problem in investigating such a system is to obtain reproducjbility on estahljshink ihe 0 - 1 0 0 ~scale for each cuan ohservable species which is proportional to some complex vette approached 1090variability with some student data. or intermediate. The poor optical properties of sultone I and ~
~
930
~~
Journal of Chemical Education
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~
~~~
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enzyme led us to use proflavin, a strict competitive inhibitor of a-Ct which has often been used to monitor a-Ct kinetics (7,9). One particularly useful experiment using proflavin was descrihed in.rHls . ~ O ~ J R N Ahv L Kantrowitz and Eisele and the difference spectrum protocol we use is taken, with slight modification, from their report (5).The reader is therefore referred t o Kantrowitz and Eisele for discussion of the difference spectrum experiment. The type I kinetic experiment is designed to approximate roughly single turnover of enzyme with sultone I, and proflavin is present in low concentration so as to detect changes in the available free enzyme concentration. The ratio of concentrations in the experiment which reflect this design is [El = [S] > [proflavin]. The data of Fieure 1are characteristic of burst kinetics in which an intermidiate builds up during the course of the reaction and is lacer hydrolyzed (5).The 465 nm ahsorhance decrease clearly shows that proflavin is heing displaced hy sultone hindine with a-Ct and that the intermediate is maximally formedafter 6 7 min. The subsequent slow increase in 465 nm absorbance indicates that sultone is decomposing, leaving more enzyme to bind to proflavin.2 This rather slow formation and degradation of sulfonylenzyme suggests the possihility of trapping theEA compound as described by the t .w.e I1 experiment. In the 80% of the enzyme becomes sulfonylated in the steady state leaving about 2&25% enzyme in the form of free enzyme and ES complex. Since the sultone is present in near saturating amounts, the 20% remaining enzyme is essentially in the ES form and an estimate of the ES = EA equilihrium, k2 4, is obtained. We have focused thus far on the instantaneous absorbance changes in Figure 2 to detect EA. But if the ahsorhance change in the assay is followed with time, a rate process is observed which demonstrates recovery of enzyme able to bmd proflavin (6a).Clearly, the EA compound detected by the instantaneous absorbance change decomposes to free enzyme and, as predicted from the mechanism (eon. should be . . (1)). . . . . the orocess . first order with an ohnerved first order rate constant equivalent to k-9 + k?. Fimre 3 eives a Gueeenheim olot of the first order dataof line dof Figure 2 with ohserveh rate constant of 0.18 min-I (64). Analysis of first-order data by the usual
--
an
F i w 4. Rate of EA Complex lwmation. Data were obtained by plotting the instantaneausabwrbana, changes at 465 nm 1e.g..as in Figure 2)as a f u n c l b 01 time of incubation of t b incubation reaction mixture. The data given were Wined by a pair of sMants using Wee separate incubanon reaction mimes. Odata from Figure 2 repiotted. Odata from two other incubation reaction
mixtures.
(absorbance at infinite time) method is certainly pwihle and ran he used in place of the Guaaenheim plot. We found from other work that k-2 is several fold great& than k3 so that the decomposition of EA nrweeds primarilvtoward recvclization of the &lfonyl moiety as opposed to h$drolysis of EA @a). This set of experiments gives students experience in interpretation of data in terms of a proposed mechanism, estimation of an equilibrium constant, evaluation of a first order rate constant, use of difference spectra, and conceptualizing molecular transformations. In our experience, poor quality data are ohtained only when temperature control i s not carefully maintained, the cuvettes are not well stoppered, or there is inadeouate mixine of solutions. Several useful variatiois of this experiment focus on the covalent sulfonvl-enzvme intermediate. I t is oossible to stabilize the intermediate by rapid titration of i h e incubation mixture (see . tvoe -. I1 experiment). to nH . 3 and then seoarate excess sultone, reaction product, and acetonitrile from the protein bv elution on a G-25 seohadex column with DH 3 HCI 16b). hek kinetics for EA decomposition can then de studied as a function of pH using the proflavin displacement assay as in Figure 2d by simply shifting from pH 3 to the desired pH. The reaction can also be observed bv followine the recoverv of catalytically competent enzyme &th satur&ing amounts of a specific substrate (6a). Acknowledgment The assays described here were developed in work supported by NIH GM22300. Literature Clted
PdH.C..aodGramJ.L., J. ~.m~c,60,14%151 Hurlbut, J.A,Bishop,C.V.,Brittain,P.C.,andPrsheim,C. W., J . CHEM. EDUC..~~,
(1) HulbuZ J. A.Ball.T.N.,
(1973).
(2)
(0 Bender, M.L., Kndy, F. J., and Wadles F.C.,J. CmM. D U C , 44.84 (1967) (5) Kantrrwitz, E. %and Eiele, G.,J. CW. Duc.,52,410 (1915). (64 lebieh, E., and Rolen. D. W.,Biwrgonie Chom., In, 11a132 (1981). (Gbl Izbicka.E.. and Bolen. D. W..Biooraonic Chem. 11).11%141 119811. , ~ ~
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( T ) ' ~ e m h a r d . ~ A , k , ~ : F . . a n d ~ a a h ; i a H., n . ZJ . MolBiol.. 18,4OM20(1966). (8) Izbicks,E.,andBolen,D.W.. J.Ampr. Chsm. Sm., IW.7625 (1978). (9) Brand% K. G.. Himoe,A.,andHam G. P., J Bid. Cham., 241,3973 (1967). (10) Gu~geoheim,E. A,, Phil. Mog.2.538 (1926). (11) Eapenson. J. H.,"Chemical Kinetics and Reaction Meebaniams,"MCCraw-Hill. New Yark, 1981, p. 25.
react with EA to we have recernlv found that nroflavin can s~ow~v -.. ,~ form free enzyme a& isuilon~lproflavinderivativs.Theeffectofthis 7~
~
~
reaction becomes apparent in the recovery phase (slow increase in
465 nm absorbance)of Figure 1. Differencespectra taken during this
phase illustrate the appearance of a new species. Further confirmation c a n be found with Silica Gel thin layer chromatography of the sample mixture by eluting with 90:lO propan0l:water (vlv) mixture and monitoring fluorescence under UV light. Volume 61 Number 10 October 1984
931
