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 60%. Active-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 tRNA 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 experiments using different ProRS preparations.

The procedure that we use was developed by Fersht et al. (1975). Basically, the assay measures the loss of ATP as a function of reaction time. As shown in equation 1, ATP and amino acid bind to the synthetase enzyme and react to form the aminoadenylate (pro-AMP in the present case). Inorganic pyrophosphatase is included in the reaction mixture to degrade pyrophosphate and prevent the reverse reaction. Therefore, further consumption of ATP will only occur when pro-AMP dissociates from the enzyme.

proline + ATP ‹--› pro - AMP + PPi (1)

The extent of reaction is quantified by measuring unreacted [-32P] ATP in a filter-binding assay. CPM's are converted to [ATP] using the specific activity of the [[-32P] ATP in the reaction mixture. The plots of [ATP] 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-AMP. A linear least squares fit to the slow phase reaction is extrapolated to zero time. Subtraction of this value from the initial [ATP] allows calculation of the active-site concentration of the enzyme. The reliability of the results depend on the amount of ATP being known to the highest accuracy possible (260 = .15,400 M-l). Still, the assay is not considered extremely precise and it is recommended to perform it in triplicate. Furthermore, the applicability of this assay is valid only if the dissociation constant of the aminoadenylate complex is << the association constant of the amino acid and ATP.

The protocol of Fersht et al. (1975) 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 mixture is calculated from the total number of samples (3), the number of time points in each assay (12), and the size of the aliquots removed per time point (10 uL) Extra reaction mixture can be made to allow for the accumulative effects of pipetting errors. Generally, 400 uL per triplicate sample will suffice.

Reaction cocktain [Stock] vol. for 400 uL144 mM Tris-Cl (pH 8.0) 1 M 57.6 uL

5.0 uM ATP 0.5 mM 4.0 uL1 mM Proline 0.1 M 4.0 uL10 mM MgCl2 1 M 4.0 uL

5 Units/mL PPiase 0.14 U/uL (2mg/mL) 14.3 uL[-32P] ATP see below 1-4 uL

Millipore H2O 312-315 uLTotal 400 uL

The amount of [-32P] ATP used depends on the age of the stock solution. Generally, 3-4 uCi total radioactivity per triplicate sample will give about 100,000 cpm in the t=0 time points. The amount of Millipore water used will make the total volume 400 uL.

133 uL of the reaction cocktail (room temp) is divided into three reaction vials. Before addition of the enzyme, 10 uL cocktail is removed and added to stop solution. The quenched samples are vortexed and placed on ice. Because of the importance of the t=0 time points, these are performed in triplicate. The stop solution consists of 700 uL 15% perchloric acid (HCIO4) and 300 uL of 3% activated charcoal suspension kept at 4oC until use. The amount of enzyme used is critical. It must be less than the initial [ATP], but close enough to it to give as large a decrease in the CPM's as possible (to maximize precision). I have found that a 1.5 fold excess of total protein (from Bio-Rad protein assay) gives an approximate active-site concentration of 4.5 uM (assuming 60% active site/total-protein ratio). At t=0, the enzyme is added to each reaction vial (103 uL) in 20 second staggers. At t=1,2,3,4,5,6,8,10 and 12 minutes, 10 uL of the assay mixture is removed and quenched as for the t=O time points. The activated charcoal binds the ATP, but not phosphate. The quenched samples are vacuum filtered on glass circle pads (Schleicher & Schuell # 30).

A new procedure used to wash out the [-32P] 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 10 ml with Millipore 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. Absolute 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 5 ml of scintillation fluid and counted for 1 minute each.

The specific activity of the [-32P] ATP in the reaction mixtures is calculated as follows: The CPM's of the t=0 time points are averaged, and divided by the total amount of ATP present in the reaction mixture. A volume correction is made for the t>0 time points to account for the dilution caused by addition of enzyme, usually ~ 7%. The CPM's of all of the samples are converted to nmol by division of the specific activity. Sample

calculations are given below. A plot of pmol ATP vs. reaction time produces a typical biphasic curve (Fig 1). The slow phase is fit by linear least squares analysis. The y-intercept is subtracted from the nmol of ATP at t=O. This is the amount of ATP that bound in the fast phase and is equal to the number of active sites in the reaction mixture. Division of this number by the volume of the sample aliquots produces the active-site concentration.

Sample Calculations:

Typical data for one trialReaction time (min) CPM pmol

0 121139 54.10 125266 55.90 120780 53.91 32645 15.82 27283 13.23 27397 13.34 26487 12.95 31383 14.76 29644 14.48 27482 13.310 25074 12.212 23302 11.3

Average CPM for t=0 time points: 122000 ± 2000 CPM.Specific Activity (t=0): 122000 CPM (5 uM ATP)-1(10 uL)-1= 2240 CPM/pmol Specific Activity (t>0): 2240 CPM/pmol (103 uL)(112.1)-1 = 2060 CPM/pmoly-int calculated from t=2 to t=12 minutes: 14.4 pmolpmol active-site in sample: 54.6 pmol (avg.) - 14.4 pmol = 40.2 pmolActive-site concentration (sample): 40.2 pmol (10 uL)-1 = 4.0 uM ProRSActive-site concentration (stock): 4.0 uM (121.1 uL) (9.1 uL)-1 = 53.2 uM

Active-Site Titration Calculations

1. Avg of the 3 t=0 points/ [ATP] (10 uL aliquot)= cpm/ pmol (S.A)

2. Adjustment for dilution (new S.A)(Cpm/pmol)X(volume after 3 t=0 are taken out)

volume after dilution with enzyme

3. Find pmol at t=0 and at y-int(Avg at t=0)/ (cpm/pmol)= pmol at t=0y-int/ (new cpm/pmol)= pmol at y-int

4. pmol= (pmol at t=0)-(pmol at y-int)

5. [Active site]sample= pmol/10 uL aliquot = uM of ProRS

6. [Active site]stockc= ( uM ProRS(sample))X(dilution volume)volume of enzyme used

7. % activity = % activity = [Active site]stock/ [ProRS]stock from Biorad