Active-site Titration (Burst assay) Protocol

The purpose of this assay is to determine the concentration of substrate binding sites in a ProRS enzyme preparation. The utility of this information is two-fold. The first is to quantitate the fraction of active enzyme contained in the total protein. Typically, this ratio is about !". #ctive-site to total protein ratios below this value might indicate needed improvement in the procedures or techniques used to isolate and purify the ProRS enzyme. The second application of the active-site concentration is in the determination of the relative catalytic activity of ProRS, such as when using modified tR$# substrates in the aminoacylation assay. The use of the active-site concentration, instead of total protein concentration, provides a control for comparison of results from e%periments using different ProRS preparations. The procedure that we use was developed by &ersht et al. '()*+,. -asically, the assay measures the loss of #TP as a function of reaction time. #s shown in equation (, #TP and amino acid bind to the synthetase enzyme and react to form the aminoadenylate 'pro#.P in the present case,. /norganic pyrophosphatase is included in the reaction mi%ture to degrade pyrophosphate and prevent the reverse reaction. Therefore, further consumption of #TP will only occur when pro-#.P dissociates from the enzyme. proline 0 #TP 1--2 pro - #.P 0 PPi '(,

The e%tent of reaction is quantified by measuring unreacted 3-45P6 #TP in a filterbinding assay. 7P.8s are converted to 3#TP6 using the specific activity of the 33 -45P6 #TP in the reaction mi%ture. The plots of 3#TP6 vs. reaction time produce a biphasic curve representative of the rapid reaction from the initial binding event and the subsequent slower reaction rate controlled by the dissociation of pro-#.P. # linear least squares fit to the slow phase reaction is e%trapolated to zero time. Subtraction of this value from the initial 3#TP6 allows calculation of the active-site concentration of the enzyme. The reliability of the results depend on the amount of #TP being 9nown to the highest accuracy possible '5 ! : .(+,;!! .-l,. Still, the assay is not considered e%tremely precise and it is recommended to perform it in triplicate. &urthermore, the applicability of this assay is valid only if the dissociation constant of the aminoadenylate comple% is << the association constant of the amino acid and #TP. The protocol of &ersht et al. '()*+, has been slightly modified and the procedure that has given us very reproducible results is detailed below= This procedure is scaled to one sample done in triplicate. The volume of the reaction mi%ture is calculated from the total number of samples '4,, the number of time points in each assay '(5,, and the size of the aliquots removed per time point '(! u>, ?%tra reaction mi%ture can be made to allow for the accumulative effects of pipetting errors. @enerally, ;!! u> per triplicate sample will suffice.

Reaction coc9tain (;; m. Tris-7l 'pA B.!, +.! u. #TP ( m. Proline (! m. .g7l5 + CnitsDm> PPiase 3-45P6 #TP .illipore A5E Total

3Stoc96 (. !.+ m. !.( . (. !.(; CDu> '5mgDm>, see below

vol. for ;!! u> +*. u> ;.! u> ;.! u> ;.! u> (;.4 u> (-; u> 4(5-4(+ u> ;!! u>

The amount of 3-45P6 #TP used depends on the age of the stoc9 solution. @enerally, 4-; u7i total radioactivity per triplicate sample will give about (!!,!!! cpm in the t:! time points. The amount of .illipore water used will ma9e the total volume ;!! u>. (44 u> of the reaction coc9tail 'room temp, is divided into three reaction vials. -efore addition of the enzyme, (! u> coc9tail is removed and added to stop solution. The quenched samples are vorte%ed and placed on ice. -ecause of the importance of the t:! time points, these are performed in triplicate. The stop solution consists of *!! u> (+" perchloric acid 'A7/E;, and 4!! u> of 4" activated charcoal suspension 9ept at ; o7 until use. The amount of enzyme used is critical. /t must be less than the initial 3#TP6, but close enough to it to give as large a decrease in the 7P.8s as possible 'to ma%imize precision,. / have found that a (.+ fold e%cess of total protein 'from -io-Rad protein assay, gives an appro%imate active-site concentration of ;.+ u. 'assuming !" active siteDtotal-protein ratio,. #t t:!, the enzyme is added to each reaction vial '(!4 u>, in 5! second staggers. #t t:(,5,4,;,+, ,B,(! and (5 minutes, (! u> of the assay mi%ture is removed and quenched as for the t:E time points. The activated charcoal binds the #TP, but not phosphate. The quenched samples are vacuum filtered on glass circle pads 'Schleicher F Schuell G 4!,. # new procedure used to wash out the 3 -45P6 phosphate was found to improve the reproducibility of this assay tremendously. This consisted of distributing the stop solution evenly on the pads and using two washes of (! ml with .illipore water by the draw down method. The draw down method removes the vacuum before addition of water, then applies it suddenly so that the liquid is drawn through the charcoal pads in a uniform manner. Simply applying the water with a squirt bottle does not give comparable results. The pads are then allowed to dry under the vacuum. #bsolute dryness is usually not attainable in a short time period, and does not seem to be essential for linear results. The pads are then placed in + ml of scintillation fluid and counted for ( minute each. The specific activity of the 3-45P6 #TP in the reaction mi%tures is calculated as follows= The 7P.8s of the t:! time points are averaged, and divided by the total amount of #TP present in the reaction mi%ture. # volume correction is made for the tH! time points to account for the dilution caused by addition of enzyme, usually I *". The 7P.8s of all of the samples are converted to nmol by division of the specific activity. Sample

calculations are given below. # plot of pmol #TP vs. reaction time produces a typical biphasic curve '&ig (,. The slow phase is fit by linear least squares analysis. The yintercept is subtracted from the nmol of #TP at t:E. This is the amount of #TP that bound in the fast phase and is equal to the number of active sites in the reaction mi%ture. Jivision of this number by the volume of the sample aliquots produces the active-site concentration. Sample 7alculations= Reaction time 'min, ! ! ! ( 5 4 ; + B (! (5 Typical data for one trial 7P. (5((4) (5+5 (5!*B! 45 ;+ 5*5B4 5*4)* 5 ;B* 4(4B4 5) ;; 5*;B5 5+!*; 544!5 pmol +;.( ++.) +4.) (+.B (4.5 (4.4 (5.) (;.* (;.; (4.4 (5.5 ((.4

#verage 7P. for t:! time points= (55!!! K 5!!! 7P.. Specific #ctivity 't:!,= (55!!! 7P. '+ u. #TP,-('(! u>,-(: 55;! 7P.Dpmol Specific #ctivity 'tH!,= 55;! 7P.Dpmol '(!4 u>,'((5.(,-( : 5! ! 7P.Dpmol y-int calculated from t:5 to t:(5 minutes= (;.; pmol pmol active-site in sample= +;. pmol 'avg., - (;.; pmol : ;!.5 pmol #ctive-site concentration 'sample,= ;!.5 pmol '(! u>,-( : ;.! u. ProRS #ctive-site concentration 'stoc9,= ;.! u. '(5(.( u>, ').( u>,-( : +4.5 u.

Active-Site Titration Calculations (. #vg of the 4 t:! pointsD 3#TP6 '(! u> aliquot,: cpmD pmol 'S.#, 5. #dLustment for dilution 'new S.#, '7pmDpmol,M'volume after 4 t:! are ta9en out, volume after dilution with enzyme 4. &ind pmol at t:! and at y-int '#vg at t:!,D 'cpmDpmol,: pmol at t:! y-intD 'new cpmDpmol,: pmol at y-int 4. pmol: 'pmol at t:!,-'pmol at y-int, 5. 3#ctive site6sample: pmolD(! u> aliquot : u. of ProRS

. 3#ctive site6stoc9c: ' u. ProRS'sample,,M'dilution volume, volume of enzyme used *. " activity : " activity : 3#ctive site6stoc9D 3ProRS6stoc9 from -iorad