Detecting ricin: Sensitive luminescent assay for ricin A-chain ribosome depurination kinetics

Article abstract of DOI:10.1021/ac8026433

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single ad

Full text of DOI:10.1021/ac8026433

Anal. Chem. 2009, 81, 2847–2853
Detecting Ricin: Sensitive Luminescent Assay for
Ricin A-Chain Ribosome Depurination Kinetics
Matthew B. Sturm and Vern L. Schramm*
Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue,
Bronx, New York 10461
Ricin is a family member of the lethal ribosome-inactivat-
ing proteins (RIP) found in plants. Ricin toxin A-chain
(RTA) from castor beans catalyzes the hydrolytic depuri-
nation of a single base from a GAGA tetraloop of eukary-
otic rRNA to release a single adenine from the sarcin-ricin
loop (SRL). Protein synthesis is inhibited by loss of the
elongation factor binding site resulting in cell death. We
report a sensitive coupled assay for the measurement of
adenine released from ribosomes or small stem-loop
RNAs by RTA catalysis. Adenine phosphoribosyl trans-
ferase (APRTase) and pyruvate orthophosphate dikinase
(PPDK) convert adenine to ATP for quantitation by firefly
luciferase. The resulting AMP is cycled to ATP to give
sustained luminescence proportional to adenine concen-
tration. Subpicomole adenine quantitation permits the
action of RTA on eukaryotic ribosomes to be followed in
continuous, high-throughput assays. Facile analysis of
RIP catalytic activity will have applications in plant toxin
detection, inhibitor screens, mechanistic analysis of depu-
rinating agents on oligonucleotides and intact ribosomes,
and in cancer immunochemotherapy. Kinetic analysis of
the catalytic action of RTA on rabbit reticulocyte 80S
ribosomes establishes a catalytic efficiency of 2.6 × 108
catalyzes the hydrolytic depurination of A4324, the first adenosine
of the conserved GAGA loop portion of the sarcin-ricin loop
(SRL).6 Depurination of the SRL causes loss of elongation factor
binding, inhibition of protein synthesis, and cellular death.
The sensitive detection of adenine is essential to establish the
catalytic mechanism of RTA, screening inhibitor libraries against
RTA action using intact ribosomes, and detection of ricin catalytic
activity in unknown samples. Current methods of quantifying
adenine from RIP depurination reactions include separation of
adenine by high-performance liquid chromatography (HPLC)
(with or without fluorescent derivatization) and a continuous
colorimetric enzyme coupled assay.7-9 These methods lack the
sensitivity to measure the continuous rates of ribosome depuri-
nation by RIPs. Immunochemistry methods for ricin detect both
the active and denatured enzyme.10,11 A gel-resolved RNA frag-
ment method using [32P]-radiolabeled ribosomes and aniline
,13
digestion at depurination sites is sensitive but cumbersome.12
We report an enzymatically coupled assay with sufficient
sensitivity to continuously measure a single adenine release from
nanomolar concentrations of intact eukaryotic ribosomes. Fem-
tomoles of ricin can be detected in minutes. The conversion of
adenine to AMP by adenine phosphoribosyl transferase (APRTase,
EC 2.4.2.7) has been reported as the first step in an adenine
colorimetric assay for the detection of RIP activity with nanomole
sensitivity for adenine.7 The measurement of AMP with subfem-
tomole sensitivity employs the pyruvate orthophosphate dikinase
(PPDK, EC 2.7.9.1) cycling reaction with firefly luciferase.14 Our
assay combines APRTase and PPDK in either coupled or discon-
tinuous reactions where free adenine is converted to AMP with
APRTase and then to adenosine 5′-triphosphate (ATP) with PPDK.
The ATP generates light via firefly luciferase and regenerates
M
-1 s-1, a diffusion limited reaction indicating catalytic
perfection even with large reactants.
Ricin from castor beans is among the most potent toxins and
is a Category B bioterrorism threat. It is toxic by inhalation, oral,
and intravenous exposure.1 The ricin type II ribosome inactivating
protein (RIP) is comprised of a catalytic A-chain (ricin A-chain,
RTA) and lectin B-chain linked by a single disulfide bond. Ricin
entry into cells is mediated by lectin B-chain binding to cell surface
galactose receptors.2 Following endocytosis, the toxin undergoes
retrograde transport from the golgi to the endoplasmic reticulum
where disulfide bond cleavage occurs and RTA is transferred to
the cytosol in a chaperone-dependent process.3-5 In the cytosol,
RTA binds the 28S portion of the 60S ribosomal subunit and
(5) Slominska-Wojewodzka, M.; Gregers, T. F.; Wa¨lchli, S.; Sandvig, K. Mol.
Biol. Cell 2006, 17, 1664–1675
(6) Endo, Y.; Mitsui, K.; Motizuki, M.; Tsurugi, K. J. Biol. Chem. 1987, 262,
5908–5912
(7) Heisler, I.; Keller, J.; Tauber, R.; Sutherland, M.; Fuchs, H. Anal. Biochem.
2002, 302, 114–122
(8) Chen, X. Y.; Link, T. M.; Schramm, V. L. Biochemistry 1998, 37, 11605–
11613
(9) Barbieri, L.; Valbonesi, P.; Bonora, E.; Gorini, P.; Bolognesi, A.; Stirpe, F.
Nucleic Acids Res. 1997, 25, 518–522
(10) Becher, F.; Duriez, E.; Volland, H.; Tabet, J. C.; Ezan, E. Anal. Chem. 2007,
79, 659–665
(11) Poli, M. A.; Rivera, V. R.; Hewetson, J. F.; Merrill, G. A. Toxicon 1994, 32,
1371–1377
(12) Olsnes, S.; Fernandez-Puentes, C.; Carrasco, L.; Vazquez, D. Eur. J. Biochem.
1975, 60, 281–288
(13) Endo, Y.; Tsurugi, K. J. Biol. Chem. 1988, 263, 8735–8739
(14) Sakakibara, T.; Murakami, S.; Eisaki, N.; Nakajima, M.-o.; Imai, K. Anal.
Biochem. 1999, 268, 94–101
.
.
.
.
* To whom correspondence should be addressed. E-mail: vern@aecom.yu.edu.
Phone: (718) 430-2813. Fax: (718)430-8565.
(1) Franz, D. R.; Jaax, N. K. In Medical Aspects of Chemical and Biological
Warfare; Sidell, F. R., Takafuji, E. T., Franz, D. R., Eds.; Textbook of Military
Medicine; Office of the Surgeon General, Department of the Army, United
States of America: Washington, DC, 1997; pp 631-642.
.
.
.
(2) Nicolson, G. L.; Blaustein, J.; Etzler, M. E. Biochemistry 1974, 13, 196–
.
204
.
.
(3) Sandvig, K.; vanDeurs, B. Physiol. Rev. 1996, 76, 949–966
(4) Wesche, J.; Rapak, A.; Olsnes, S. J. Biol. Chem. 1999, 274, 34443–34449
.
.
.
10.1021/ac8026433 CCC: $40.75 2009 American Chemical Society
Published on Web 03/20/2009
Analytical Chemistry, Vol. 81, No. 8, April 15, 2009 2847

Figure 1. Adenine released from the ricin A-chain (RTA) depurination of stem-loop (A-10) or ribosome (60S SRL) is converted to AMP with
adenine phosphoribosyl transferase (APRTase) and then to ATP with pyruvate orthophosphate dikinase (PPDK). ATP drives the luciferase
enzymatic reaction to produce luminescence and AMP to be recycled to ATP. At neutral pH, all reactions can be combined to measure adenine
continuously from ribosomes.
AMP, which is rapidly converted to ATP by PPDK (Figure 1).
Continuous measurements are accomplished in a 96-well plate
format by combining the luciferase reagent with APRTase/PPDK
(adenine to ATP) coupling enzymes.
autoclave treatment) was used for all enzymatic reactions. All other
reagents used were purchased in the highest purity available from
Fisher Scientific (Pittsburgh, PA) or Aldrich Chemical Corp.
(Ashland, MA). Spectrophotometric assays and concentrations of
adenine, ribosomes, and oligonucleotides were measured using
a Varian Cary 100 diode array spectrophotometer. Luminescence
was measured on a GloMax 96-well luminometer from Promega
(Madison, WI).
The depurination of 80S rabbit reticulocyte ribosomes and 60S
yeast ribosomes by RTA was investigated using the adenine-
luciferase coupled assay to establish the initial rate kinetic
parameters benchmarked in both continuous and discontinuous
assay formats. A-10, an RNA stem-loop (5′-CGCGAGAGCG-3′)
mimic of the SRL, was assayed at pH 4.0 with RTA to establish
its kinetic parameters for comparison with kinetic results from
an HPLC assay.8 A previously uncharacterized RNA/DNA hybrid
stem-loop A-14 2-dA (5′-CGCGCGdAGAGCGCG-3′, where dA is
2′-deoxy-AMP in 3′,5′-linkage) was also assayed to characterize a
high-efficiency synthetic substrate for routine detection and kinetic
analysis of RIP activity.15
Adenine to ATP Conversion Buffer. A volume of 50 mL of
enzyme buffer was prepared with 100 mM Tris-acetate pH 7.7, 2
mM phosphoenolpyruvic acid, 2 mM sodium pyrophosphate, 2
mM 5-phospho-R-D-ribosyl-1-pyrophosphate (PRPP), 15 mM
NH4SO4, and 15 mM (NH4)2MoO4 in DEPC treated water with
phosphatase inhibitors (Roche PhosSTOP, 5 tablets). Charcoal/
cellulose (1:5) powder was suspended in DEPC water and
packed into a PD10 column with 100 mL of water at 2 mL/
min. The enzyme buffer was passed through the column at 2
mL/min to remove adenine, AMP, and ATP contamination. The
eluate was filtered (0.2 µm) and stored in 1 mL aliquots at -80
°C. Prior to use in adenine assays, 2× coupling buffer was
prepared by adding 10 mM MgSO4, 4 units of APRTase, 8 units
of PPDK, and 1 µL of SuperRNasin (Ambion) per 1 mL of
enzyme buffer. One unit of enzyme activity was defined as the
amount which converts 1 µmol of substrate per minute at 20
°C. The enzymatic activity of PPDK was determined spectro-
photometrically at 340 nM by coupling pyruvate formation to
lactate dehydrogenase with 0.2 mM reduced nicotinamide
adenine dinucleotide (NADH) and 50 µM AMP added to start
the reaction at 20 °C in a buffer (500 µL of 2× coupling buffer
added to 500 µL H2O with 10 mM MgSO4 and 1 µL SuperR-
Nasin (Ambion). APRTase activity was determined in similar
conditions without AMP but with excess PPDK and 50 µM of
adenine to start the reaction.
MATERIALS AND METHODS
Ricin toxin A-chain (RTA) was purchased from Sigma. Ricin
(RCA60) from Vector Laboratories (Burlingame, CA) was a
generous gift from Dr. Pamela Stanley (Albert Einstein College
of Medicine). APRTase was the generous gift of Dr. Minkui Luo
(Albert Einstein College of Medicine). The plasmid containing
the PPDK clone from Clostridium symbiosum was a generous gift
provided by Dr. Debra Dunaway-Mariano (University of New
Mexico). 60S Yeast ribosomes were the generous gift from Dr.
Uma Maitra (Albert Einstein College of Medicine). Rabbit reticu-
locyte lysate (untreated, $100) was purchased from Promega
(Madison, WI) and is sufficient for ∼100 assays as described
below. Oligonucleotides A-10 and A-14 2-dA were purchased from
Dharmacon (Lafayette, CO). Sufficient A-12 2-dA for 5 000 assays
is currently $300. Firefly luciferase ATP assay kit (ATPlite) was
purchased from Perkin-Elmer (Waltham, Massachusetts). Phos-
phatase inhibitors (PhosSTOP) were purchased from Roche
Applied Science (Indianapolis, IN). RNase inhibitor (SuperRNasin)
was purchased from Ambion (Austin, TX). Buffers and enzyme
preparations were checked for RNase activity using the RNaseA-
lert kit from Ambion (Austin, TX). Diethylpyrocarbonate (DEPC)
treated water (0.1% DEPC stirred for 20 min followed by 30 min
Expression and Purification of S. cerevisiae APRTase
(scAPRTase). scAPRTase (N-terminal 6× His) was expressed and
purified as described previously with some modification.16 The
>95% pure APRTase by sodium dodecylsufate-polyacrylamide gel
electrophoresis (SDS-PAGE) was reconstituted with 10% glycerol
(final volume ratio), concentrated, and then purified on a Super-
(16) Shi, W.; Tanaka, K. S. E.; Crother, T. R.; Taylor, M. W.; Almo, S. C.;
(15) Amukele, T. K.; Schramm, V. L. Biochemistry 2004, 43, 4913–4922
2848 Analytical Chemistry, Vol. 81, No. 8, April 15, 2009
.
Schramm, V. L. Biochemistry 2001, 40, 10800–10809.

dex75 (GE Science) in 50 mM Tris-HCl pH 7.4, 20 mM KCl, 5
mM MgCl2 containing 10% glycerol. The APRTase fractions
were concentrated to ∼10 mg/mL and flash frozen in dry ice/
ethanol and stored at -80 °C. The gel filtration purification
step was needed to remove trace DNAase/RNAase activities
in scAPRTase.
was calculated from a standard plot of luminescence (RLU) versus
varying picomoles of known adenine in identical sample buffer
conditions.
For continuous assays of adenine, a 2× continuous assay buffer
was prepared by adding 200 µL of D-luciferin/luciferase (ATPlite)
to 1 mL of 2× coupling buffer. The 2× continuous assay buffer
was equilibrated to room temperature (20 °C) for 10 min prior to
assays. A volume of 25 µL of 2× continuous assay buffer was added
to 25 µL of sample in a 96-well plate followed by 2 min of
continuous luminescent (RLU) acquisition on a luminometer.
Adenine content was calculated from a standard calibration curve
in identical sample buffer conditions as described above.
In both assay formats, contamination of AMP and ATP was
evaluated in control reactions with sample by excluding APRTase
or APRTase/PPDK, respectively, in the 2× coupling/continuous
assay buffers (Figure 1).
Discontinuous Assay Kinetics. Varying concentrations of
substrate A-10 or A-14 2-dA were incubated at 37 °C for 5 min in
acidic RTA buffer (10 mM potassium citrate-KOH and 1 mM
EDTA at pH 4.0). Reactions were initiated by the addition of RTA
at concentrations typically between 0.5 and 20 nM to a total
reaction volume of 25 µL. The reactions were quenched to neutral
pH at timed intervals with 25 µL of 2× coupling buffer to give
<15% product formation. In parallel, five adenine standards and
three controls (buffer, buffer + substrate, and RTA + buffer) were
made in acidic RTA buffer diluted with 2× coupling buffer in
volumes identical to the substrate assay condition. The quenched
RTA reactions, standards, and control solutions were incubated
at room temperature for at least 2 min (to convert adenine to ATP).
Aliquots (20 µL) were assayed for picomoles of ATP as described.
Luminescence (RLU) was converted to picomoles of adenine from
the adenine standard curve fit (corrected for background from
controls), and initial rate kinetics were fit to the Michaelis-Menten
equation for the calculation of substrate kcat and Km.
For discontinuous ribosome assays, 60S or 80S ribosome were
incubated for 5 min in pH 7.4 ribosome reaction buffer at 20 °C
(a temperature identical to continuous assays). Reactions were
initiated by the addition of RTA at concentrations between 30 and
70 pM in a total reaction volume of 25 µL. The reactions were
quenched at timed intervals to give <15% product formation with
60 mM HCl, incubated on ice for 2 min, and brought back to
neutrality with 60 mM KOH. An equal volume of 2× coupling
buffer was added to the quenched reactions and incubated at room
temperature for at least 2 min. Adenine standards and controls
(as described for stem-loop assays) were prepared in ribosome
reaction buffer and were quenched in an identical buffer condition
as the ribosome RTA catalytic reactions. Aliquots (20 µL) were
assayed for picomoles of ATP as described. Ribosome concentra-
tion was determined by depurinating (to completion) two stock
concentrations with 500 nM RTA and comparing the final
luminescence to the adenine standard curve fit. Picomoles of
adenine released during the assay and initial rate kinetics were
calculated identically to stem-loop assays after appropriate back-
ground corrections were made from the controls. Background
degradation of any of the RNA substrates is not significant when
standard RNA handling procedures are followed.
Expression and Purification of Clostridium symbiosum
PPDK. PPDK was expressed and purified as described previously
with some modifications.17,18 The plasmid encoding PPDK from
Clostridium symbiosum was expressed in Escherichia coli JM101
grown in Luria broth (LB) medium with 12.5 µM tetracycline to
an A600 of 1.4. The cells were harvested by centrifugation at 2
000g for 30 min at 4 °C. The cell pellet was suspended in buffer
(20 mM imidazole, 2.5 mM sodium ethylenediaminetetraacetic
acid (Na2EDTA), 2 mM dithiothreitol (DTT), 75 mM KCl, pH
6.8 with complete protease inhibitor cocktail (Roche)) and the
cells were disrupted by French press followed by centrifugation
at 17 000g for 1 h at 4 °C. Treatment with streptomycin sulfate,
ammonium sulfate, and DEAE cellulose column purification
steps,17,18 PPDK was subsequently dialyzed in 20 mM imidazole,
2.5 mM Na2EDTA, 1 mM DTT, 88 mM KCl, pH 6.4 at 4 °C and
loaded onto a Sephacryl S-200 column (GE Science) and eluted
in dialysis buffer. Pure PPDK fractions were identified by SDS-
PAGE. Glycerol to 10% (final volume ratio) was added, and
aliquots were frozen in dry ice/ethanol and stored at -80 °C.
Ribosomes. Rabbit reticulocyte 80S ribosomes were purified
from untreated rabbit reticulocyte lysate. A volume of 500 µL of
lystate was layered onto 250 µL of sucrose cushion (1 M sucrose,
20 mM Tris-HCl pH 7.5, 500 mM KCl, 2.5 mM MgCl2, 0.1 mM
EDTA, and 0.5 mM DTT) and centrifuged at 100 000 rpm in a
TLA 120.2 rotor for 2 h at 4 °C. The glassy pellet was washed
twice on ice with ribosome storage buffer (20 mM Hepes-KOH
pH 7.7, 100 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 250 mM
sucrose, and 1 mM DTT) and suspended in a minimal volume
of the same buffer. The 80S ribosome was flash frozen in dry
ice/ethanol and stored at -80 °C. Ribosomes were stable while
frozen, but multiple freeze-thaw cycles reduces their reactivity
for the ricin assays. Once thawed, ribosome samples are stable
on ice for the several hours needed to complete reactions.
Prior to use in catalytic assays, 60S and 80S ribosome were
buffer exchanged into ribosome reaction buffer (20 mM Tris-HCl
pH 7.4, 25 mM KCl, and 5 mM MgCl2) with a G-25 microcen-
trifuge desalting spin column at 4 °C (Pierce). Concentrations
of 60S and 80S were calculated by absorbance at 260 nm.19
Active concentrations of 60S and 80S ribosome were deter-
mined by complete depurination by RTA in kinetic assays (see
Materials and Methods). The contamination of AMP and ATP
in isolated ribosomes was reduced by passing the ribosomes
through a size exclusion spin column as described above.
Assaying Samples for Adenine. In the discontinuous assay
format, 25 µL of 2× coupling buffer was added to 25 µL of sample
and incubated at room temperature for several minutes. In a 96-
well luminometer plate, 20 µL of the reaction was added to 80 µL
of D-luciferin/luciferase reagent and was assayed for ATP with a
luminometer as described by the manufacturer. Adenine content
(17) Wang, H. C.; Ciskanik, L.; Dunaway-Mariano, D.; von-der-Saal, W.; Vil-
lafranca, J. J. Biochemistry 1988, 27, 625–633
(18) Goss, N. H.; Evans, C. T.; Wood, H. G. Biochemistry 1980, 19, 5805–5809
(19) Wool, I. G. Annu. Rev. Biochem. 1979, 48, 719–754
.
Continuous Assay Kinetics. In 96-well plate format, varying
concentrations of ribosome were individually assayed in the kinetic
.
.
Analytical Chemistry, Vol. 81, No. 8, April 15, 2009 2849

Figure 2. Adenine standards: (A) plot of luminescence (RLU) versus
adenine (0.4-840 pmol) measured in the discontinuous assay format;
(B) continuous luminescent plots for increasing concentrations of
adenine measured over a 2 min period. From bottom to top are blank,
0.5, 1, 2, 4, and 8 pmol of adenine.
acquisition mode of the luminometer in a total reaction volume
of 50 µL in 1× continuous assay buffer. RTA was prepared in ice
cold ribosome reaction buffer with a single dilution from stock
and equilibrated to room temperature before assays. A 1 min blank
reading for each ribosome concentration was made before the
addition of RTA (30-70 pM) to ensure a stable luminescent signal.
Luminescence was monitored for several minutes after RTA
addition. Adenine standards in the concentration range of the
assay reactions were preformed before and after continuous
ribosome assays. Ribosome concentration was determined by
depurinating (to completion) two stock concentrations with 500
nM RTA and comparing the final luminescence to the adenine
standard curve fit. The initial rate of adenine formation from the
catalytic reactions were calculated by converting luminescent rate
(lumens/s) to enzymatic rate (picomoles of adenine/min/pico-
moles of enzyme) using the adenine standard curve fit, and kinetic
parameters kcat and Km were calculated by fitting initial rates to
the Michaelis-Menten equation.
Figure 3. Discontinuous adenine-luciferase assay format kinetic
curve fits for RTA catalysis of stem-loop RNA A-10 (A) and A-14 2-dA
(B) at pH 4.0. Kinetic curve fits for RTA catalysis of 80S rabbit (C)
and 60S yeast (D) ribosome at pH 7.4 in 20 mM tris-HCl, 25 mM
KCl, and 5 mM MgCl2.
RESULTS AND DISCUSSION
Standard Quantitation of Adenine. In the discontinuous
assay format, adenine standards between 0.4 and 840 pmol were
used to establish a standard curve and gave a linear luminescence
response when assayed with D-luciferin/luciferase reagent (Figure
2A). The conversion of adenine to ATP proceeds to chemical
completion as determined by comparing luminescence signals
from known quantities of adenine and ATP (data not shown). Both
gave equivalent light signals under the conditions of Figure 2. In
the continuous assay format, luminometer recordings of light
output versus adenine concentration showed a stable lumines-
cence signal after 2 min (Figure 2B). The Km values of adenine
for APRTase and of AMP for PPDK are both 9 µM, and several
reactions of Figure 2A contained initial adenine concentrations
Figure 4. Continuous assay measurements: (A) Initial rate slopes
(lumens/second) of increasing concentrations of 80S rabbit ribosome
with 33 pM RTA and (B) kinetic curve fit of the calculated kinetic rate
from part A versus the 80S ribosome concentration. (C) Kinetic curve
fit for RTA catalysis of 60S yeast ribosome measured in the
continuous adenine-luciferase assay.
Discontinuous Assay for Adenine Release from Stem-
Loop RNA. RTA depurinates an adenine from stem-loop RNA
and RNA/DNA hybrid substrates at pH 4.0 but not at pH 7.8,15,22
Small RNA stem-loops were assayed discontinuously at pH 4.0,
and the reaction was stopped by the addition of 2× coupling buffer
to provide a neutral pH. RTA initial rate kinetics for the catalysis
of stem-loop substrate A-10 measured by the adenine-luciferase
assay gave a saturating curve fit with a calculated kcat of 4.7 ± 0.3
min-1 and Km of 2.2 ± 0.3 µM (Figure 3A). Previously determined
kinetics for RTA catalysis using HPLC analysis of adenine release
in large scale reactions with A-10 are consistent with these values
(kcat of 4.1 min-1 and Km of ∼4.0 µM).8 With the adenine-
luciferase assay, RTA catalysis of the A-14 2-dA stem-loop RNA/
DNA hybrid was measured to give a kcat of 1110 ± 50 min-1
,20
lower than these Km values.16 The coupling enzymes were in
sufficient excess to reach full conversion to ATP in less than 2
min, even with subsaturating adenine. Firefly luciferase has two
Km values for ATP, 111 µM for the initial flash and 20 µM for
the continuous but lower production of light.21 ATP concentra-
tions of the aliquots assayed were below the initial flash and
continuous light production Km values for the detection system.
The linear response to adenine (ATP) in Figure 2A supports
light production from the high Km process, resulting in a
functionally linear response.
(20) Ye, D.; Wei, M.; McGuire, M.; Huang, K.; Kapadia, G.; Herzberg, O.; Martin,
B. M.; Dunaway-Mariano, D. J. Biol. Chem. 2001, 276, 37630–37639
(21) DeLuca, M.; McElroy, W. D. Biochem. Biophys. Res. Commun. 1984, 123,
764–770
.
(22) Amukele, T. K.; Roday, S.; Schramm, V. L. Biochemistry 2005, 44, 4416–
.
4425.
2850 Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

Table 1. RTA Kinetic Parameters
substrate
Km
(µM)
Kcat (min-1
)
Kcat/K
(M-1s-1
)
m
A-10a b
2.2 ± 0.3
4.7 ± 0.3
1110 ± 50
460 ± 90
3.6 × 104
1.8 × 106
2.8 × 107
1.9 × 107
2.6 × 108
1.4 × 108
,
A14 2-dAa b
10.3 ± 1.6
,
60S yeast ribosomea
60S yeast ribosomec
80S rabbit ribosomea
80S rabbit ribosomec
0.270 ± 0.08
0.240 ± 0.06
0.150 ± 0.025
0.100 ± 0.005
270 ± 40
2310 ± 150
820 ± 10
a Substrate assayed in discontinuous format. b Substrate assayed at 37 °C. c Substrate assayed in continuous format.
and a Km of 10.3 ± 1.6 µM at pH 4.0 (Figure 3B). The
deoxyadenosine at the depurination site provides a better substrate
compared to RNA A-14 (a reported kcat of 219 ± 14 min-1 and Km
of 8.1 ± 0.7).8 Deoxyadenosine at the depurination site
(GdAGA) of RNA stem-loop oligonucleotides increases the
catalytic efficiency (kcat/Km) by RTA a factor of ∼4 for
catalysis.15 RTA is known to form a ribocation transition state
and incorporation of the deoxyribosyl group specifically at the
depurination site chemically destabilizes the ribosidic bond to
kinetics for RTA depurination of 60S yeast ribosomes at pH
7.4 conformed to Michaelis-Menten kinetics with a kcat of 460
± 90 min-1 and a Km of 270 ± 80 nM at 20 °C (Figure 3D).
Continuous Assay for Adenine Release from Ribosomes.
In continuous coupled assays for RTA, the conversion of adenine
to ATP must be rapid relative to the release of adenine from the
substrates. With fixed adenine concentrations, even at the highest
concentrations, steady-state light production occurred within 2 min
(Figure 2B). We considered it feasible to use continuous coupled
assays for adenine release from intact ribosomes. The RTA
catalyzed release of adenine from 80S rabbit and 60S yeast
ribosomes were measured continuously at pH 7.7 by monitoring
a linear increase in luminescence. The concentration of RTA in
continuous ribosome reaction assays was kept low to ensure that
production of adenine is rate limiting. In the continuous ribosome
assays, the concentrations of RTA was 33 and 66 pM for 80S and
60S ribosomes, respectively. The initial rates were dependent on
ribosome concentration (Figure 4A). The initial rates of catalysis
asafunctionof80SribosomeconcentrationfittotheMichaelis-Menten
equation with a kcat of 820 ± 10 min-1 and Km of 100 ± 5 nM
(Figure 4B). Under the same conditions, the kcat and Km values
for RTA catalysis of 60S yeast ribosomes were 270 ± 40 min-1
and 240 ± 60 nM, respectively (Figure 4C). RTA action on
ribosomes in continuous and discontinuous assays gave similar
Km values but a ∼2-3-fold difference in kcat (Table 1). This
difference potentially results from varying conditions between the
two assay formats, but this result was not further investigated.
80S rabbit and 60S yeast ribosomes have Km values in the range
of 100-270 nM with the tightest binding being 100 nM for
80S rabbit ribosomes under the conditions used for the
continuous assay. Previous reports of the action of RTA on 80S
rabbit reticulocyte ribosomes by aniline cleavage, [32P] end-
labeling and gel separation gave a kcat of approximately 1500
min-1 and Km of 100-200 nM depending on conditions,
completely comparable with the adenine-luciferase assays
,23,24
the adenine and thereby increases the reaction rate.15
RTA and other type I and II RIPs have a low pH catalytic
optimum on nucleic acid substrates such as stem-loop RNA,
poly(A), and herring sperm DNA, while the natural ribosome
substrate is depurinated optimally at physiologic pH.9,25 RTA
catalysis of stem-loop substrates was not detectable above pH 6.5
with either A-10 or A-14 2-dA stem-loop oligonucleotides (up to
100 µM) with 2 µM RTA for 10 min. The pH 4.0 catalytic optimum
of RTA on 6 to 18-mer stem-loop DNA and RNA 28S SRL mimic
oligonucleotides is known to result from the protonation of two
ionizable residues on substrate and/or RTA.8,15,22
Discontinuous Assay of Adenine Release from Ribo-
somes. Kinetic analysis of reactions using intact ribosomes as
substrates where the Km for ribosomes is low and a single
adenine is released from the macromolecular complex of
approximately 5 million Da presents a challenge. AMP and ATP
contamination in ribosome preparations were reduced with a
size exclusion spin column (Materials and Methods). The initial
rates of adenine release from ribosomes at pH 7.4 in a discontinu-
ous assay involved quenching the reaction followed by adenine
conversion to ATP. RTA reactions were quenched with HCl to
inactivate RTA and subsequently neutralized to pH 7.0 with KOH.
The depurination of ribosomes by RTA occurred with a stoichi-
ometry of one adenine released per ribosome. This finding
provides a unique method to quantify the chemical concentration
of eukaryotic ribosomes. With the use of an enzyme concentration
of 33 pM RTA, the initial rate kinetics for depurination of 80S rabbit
reticulocyte ribosomes gave a saturation curve with a kcat of 2310
± 150 min-1 and a Km of 150 ± 25 nM at 20 °C (Figure 3C).
This reaction has a catalytic efficiency (kcat/Km) of 2.6 × 108 M-1
(Table 1).12 The ribosomes are depurinated at catalytic rates (kcat
)
comparable to that of deoxyadenosine (GdAGA) stem-loop
oligonucleotides A-14 2-dA assayed at pH 4.0 (Table 1), but the
Km value for the interaction with ribosomes indicates tighter
binding.
We investigated if RTA hydrolyzes A-10 or A-14 2-dA stem-
loop at neutral pH in the presence of ribosomes (rabbit or yeast).
Adenine release was only from ribosome depurination. Mam-
malian ribosomal proteins L9 and L10e are adjacent to the RTA
SRL binding site and are likely to provide recognition elements
s
-1, at or near the diffusion limit for enzymatic catalysts with
slowly diffusing substrates and a 32 kDa enzyme.26 Initial rate
(23) Chen, X. Y.; Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 2000, 122,
1609–1617
(24) Chen, X. Y.; Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 2000, 122,
6527–6534
(25) Barbieri, L.; Ciani, M.; Girbe´s, T.; Liu, W.; VanDamme, E. J. M.; Peumans,
W. J.; Stirpe, F. FEBS Lett. 2004, 563, 219–222
(26) Stroppolo, M. E.; Falconi, M.; Caccuri, A. M.; Desideri, A. Cell. Mol. Life
Sci. 2001, 58, 1451–1460
.
.
.
.
Analytical Chemistry, Vol. 81, No. 8, April 15, 2009 2851

for RTA catalysis.27 Conformational changes in RTA upon ribo-
some binding have also been proposed.28 Thus, the pH-modulation
effects occur only in the ribosome-RTA complex and are not
maintained for RTA release and rebinding of small stem-loop RNAs
under physiologic conditions with ribosomes.
Catalytic Perfection in RTA Depurination. Catalytic perfec-
tion in enzymatic reactions results when every diffusional collision
between substrate and enzyme leads to product formation. In
these cases, the catalytic rate is defined by the diffusion rate of
the smaller reactant.29 Well characterized enzymes exhibiting
catalytic perfection include triose phosphate isomerase (kcat/Km
) 3.0 × 108 M-1 s-1), acetylcholinesterase (kcat/Km ) 1.4 × 108
M
-1 s-1), ketosteroid isomerase (kcat/Km ) 1.3 × 108 M-1 s-1),
and ꢀ-lactamase (kcat/Km ) 1.0 × 108 M-1 s-1).26,30 Remarkably,
the catalytic efficiency (kcat/Km) for RTA on rabbit 80S ribosomes
(2.6 × 108 M-1 s-1 for the discontinuous assay conditions and
1.4 × 108 M-1 s-1 for the continuous assay) is similar, even
though diffusion for the RTA-ribosome system is limited by
the relatively large molecular weight of RTA (Mr ) 32 kDa).
The finding that RTA has a kcat/Km value above the diffusion
limit for uncharged reactants of this size indicates that the
charge difference between RTA and the ribosome plays a
significant role in its extraordinary catalytic efficiency.
Figure 5. (A) Plot of luminescent signal-to-noise ratio (S/N ratio)
versus concentrations of ricin. Ricin concentrations were from 0.380
to 6.2 nM in the discontinuous assay format. Reaction mixtures of
25 µL contained acidic RTA buffer + 0.005% triton X-100 with 4 µM
A-14 2-dA at 37 °C for 10 min (O) and 20 min (0). Triplicate samples
gave the mean and 95% confidence interval error bars. A linear fit to
95% confidence interval data points are shown (disconnected lines).
(B) Plot of lumens (RLU) versus time (seconds) for the detection of
140 pM (4.5 ng/mL) RTA with increasing concentrations of 80S rabbit
ribosome in 50 µL reactions (bottom to top: control, 20, 40, 80, and
160 nM 80S ribosome). The continuous assay format has the
background signal subtracted from the curves. (C,D) Data points of
lumens (RLU) versus controls (buffer + RTA, buffer + 80S, 80S/
RTA no APRTase) and samples containing RTA (6.25-50 pM) with
50 nM 80S ribosome repeated in triplicate and measured for 1 s in
the continuous assay format after 5 (C) and 10 (D) min of reaction,
respectively. Error bars were calculated as 95% confidence intervals.
Ricin/RTA Detection. One application for a sensitive and
rapid quantification of adenine is to permit the detection of RTA,
ricin, and related RIPs in samples using small stable nucleic acid
constructs. Ricin holotoxin can depurinate synthetic substrates
including A-14 2-dA without reductive pretreatment to release the
catalytic A-chain (RTA).9 Truncated stem-loop substrates have a
pH optimum of 4.0 for binding and catalysis, and the two-step
discontinuous assay is optimal for this application. The RNA stem
loop A-10 is hydrolyzed at 4.7 min-1 by RTA (Table 1). On the
basis of the sensitivity of the assay here, a 50 µL assay containing
20 µM A-10 and 6.6 nM of RTA (∼200 ng/mL) would provide a
robust luminescent signal in 30 s. The more active A-14 2-dA (kcat
of ∼1110 min-1) incorporates a labile adenine in an RNA/DNA
hybrid with a 2′-deoxy ribose at the cleavage site and would
be an ideal synthetic substrate for subnanomolar RTA/ricin
detection (Table 1). In 25 µL reactions, depurination of 4 µM
A-14 2-dA with ricin concentrations as low as 400 pM (25 ng/
mL) were detected in an assay time of 20 min without reductive
pretreatment with the detection limit defined as a signal-to-noise
ratio (S/N) of 3 (Figure 5A). Ricin detection in environmental or
serum samples would need to be desalted/buffer exchanged into
acidic RTA buffer prior to detection assays to remove contaminat-
ing AMP/ATP and physiologic salts (inhibitory to the pH 4.0 RTA/
ricin catalytic reaction). Sample RNase contamination is analyzed
in control reactions by omitting APRTase in the 2× coupling buffer
and quantifying ATP formation from nuclease generated AMP
(Figures 1 and 5C,D). Thus, samples containing interfering
nuclease contamination would be identified and require applicable
commercial RNase inhibitor treatment.
80S ribosome is the most efficient RTA detection probe with
the lowest Km and fastest kcat of substrates described herein
(Table 1). A concentration of 140 pM (4.5 ng/mL) of RTA in 50
µL reactions was detected within minutes using increasing
concentrations of gel filtered 80S ribosome in the continuous
adenine-luciferase assay format (Figure 5B). In the same format
using 50 nM crude 80S ribosome substrate, the RTA detection
limit (S/N ratio of 3) was 50 pM (1.6 ng/mL) in 5 min and 25 pM
(0.8 ng/mL) in 10 min (Figure 5C,D). For the routine detection
of ricin/RTA using 80S ribosomes, reductive pretreatment of the
sample (ricin holotoxin), stringent nuclease control, and sensitive
ribosome preparations make this method less practical than the
discontinuous ricin detection assay using truncated stem-loop A-14
2-dA substrate (Figure 5A).
CONCLUSIONS
Coupling firefly luciferase to ATP formed from adenine
permitted sensitive quantification of the RTA depurination reac-
tion. Luminescence from adenine is produced from APRTase,
(27) Vater, C. A.; Bartle, L. M.; Leszyk, J. D.; Lambert, J. M.; Goldmacher, V. S.
J. Biol. Chem. 1995, 270, 12933–12940
(28) Argent, R. H.; Parrott, A. M.; Day, P. J.; Roberts, L. M.; Stockley, P. G.;
Lord, J. M.; Radford, S. E. J. Biol. Chem. 2000, 275, 9263–9269
(29) Burbaum, J. J.; Raines, R. T.; Albery, W. J.; Knowles, J. R. Biochemistry
1989, 28, 9293–9305
.
PPDK, and D-luciferin/luciferase coupling enzymes (Figure 1).
.
The assay can measure subpicomole quantities of adenine in
discontinuous and/or continuous assay formats (Figure 2). A
luminescent signal stable for several minutes is observed with an
.
(30) Fersht, A. Enzyme and Structure Mechanism; W. H. Freeman and Co.: New
York, 1985.
optimized D-luciferin/luciferase formulation along with AMP
2852 Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

cycling from PPDK and luciferase enzymes (Figure 2, Figure 1).
We envision that the buffer conditions and -luciferin/luciferase
continuous assay format. Catalytic perfection is achieved
through the combined interactions of the polycationic nature
of the RTA active site, anionic distribution of the substrate, and
potential remote contacts of RTA with adjacent SRL ribosomal
proteins. The 80S rabbit ribosome serves as a rapid continuous
detection probe for active RTA. A concentration of 140 pM (4.5
ng/mL) of RTA in 50 µL reactions was detected within minutes
by directly measuring adenine release from 80S rabbit ribo-
some (Figure 5B). The RTA detection limit using 80S ribosome
in continuous assays was 50 pM (1.6 ng/mL) in 5 min and 25 pM
(0.8 ng/mL) in 10 min (Figure 5C,D). Ricin holotoxin detection
via 80S ribosome catalysis would require reductive pretreatment
of the sample. The adenine-luciferase assay can easily be adapted
for high throughput to detect potential RTA inhibitors or the
activity based detection of other RIP enzymes to aid in isolation
and discovery.
D
preparation of the adenine-luciferase assay buffers can be further
optimized to adapt it to other applications where adenine detection
is desired. For assaying larger quantities of adenine (mM), the
adenine to ATP coupling buffer (2× coupling buffer) can be used
spectrophotometrically by coupling the pyruvate generated by
PPDK to a lactate dehygrogenase/NADH assay (Figure 1,
Materials and Methods).
RTA kinetics were investigated on truncated sarcin-ricin loop
(SRL) mimics A-10 and A-14 2-dA at acidic pH discontinuously
where the adenine-luciferase assay provided a neutral pH quench
and converted adenine to ATP (Figure 3A,B). A-14 2-dA, a hybrid
RNA/DNA stem-loop, was efficiently catalyzed (kcat/Km ) 1.8 ×
106 M-1 s-1) by RTA and could serve as a rapid detection probe
for the active toxin or access RTA immunoconjugate catalytic
activity. With the use of A-14 2-dA substrate, untreated ricin
was detected in 20 min with a limit of 400 pM (25 ng/mL)
toxin (Figure 5A).
ACKNOWLEDGMENT
This work was supported by NIH Research Grant CA072444
and NIH Training Grant GM07288.
RTA kinetics on ribosomes demonstrated low nanomolar
binding (Km ) 100 and 270 nM) for 80S rabbit reticulocyte and
60S yeast, respectively (Table 1). The catalysis of 80S ribosome
by RTA approached the diffusion rate limit for enzymatic reactions
with a catalytic efficiency (kcat/Km) of 2.6 × 108 M-1 s-1 for the
discontinuous assay conditions and 1.4 × 108 M-1 s-1 for the
Received for review October 1, 2008. Accepted February
4, 2009.
AC8026433
Analytical Chemistry, Vol. 81, No. 8, April 15, 2009 2853