In vitro site-specific incorporation of fluorescent probes into β-galactosidase

Article abstract of DOI:10.1021/ja963023f

Fluorescence spectroscopy is a powerful biophysical technique for studying protein structure, function, dynamics, and intermolecular interactions. Such studies are often conducted using intrinsic probes, such as tryptophan residues, or extrinsic probes in

Full text of DOI:10.1021/ja963023f

6
J. Am. Chem. Soc. 1997, 119, 6-11
In Vitro Site-Specific Incorporation of Fluorescent Probes into
â-Galactosidase
Lance E. Steward,† Cynthia S. Collins,† Marcella A. Gilmore,† Justin E. Carlson,‡
J. B. Alexander Ross,*,‡ and A. Richard Chamberlin*,†
Contribution from the Department of Chemistry, UniVersity of California,
IrVine, California 92697, and Department of Biochemistry, Mt. Sinai School of Medicine,
One GustaVe L. LeVy Place, New York, New York 10029
ReceiVed August 28, 1996X
Abstract: Fluorescence spectroscopy is a powerful biophysical technique for studying protein structure, function,
dynamics, and intermolecular interactions. Such studies are often conducted using intrinsic probes, such as tryptophan
residues, or extrinsic probes introduced by post-translational modification, such as dansyl. Specificity, however, is
often a concern since many proteins contain more than one tryptophan and chemical modification often will occur
at more than one site. Herein we report the in Vitro, site-specific incorporation of three fluorescent amino acid
analogues, 5-hydroxytryptophan, 7-azatryptophan, and ꢀ-dansyllysine, each of which was incorporated into
â-galactosidase at a single designated site.
Protein fluorescence spectroscopy is a powerful biophysical
solution to these limitations, allowing the site-specific introduc-
tion of a single fluorescent probe whose spectral window is
distinct from all endogenous amino acid residues. In this paper
we report the incorporation of three noncoded amino acids with
useful spectral properties into â-galactosidase: the tryptophan
analogues 5-hydroxytryptophan (5-OHTrp, 2), 7-azatryptophan
(7-azaTrp, 3), and ꢀ-dansyllysine (dnsLys, 4). Both tryptophan
technique for studying protein structure, function, dynamics, and
intermolecular interactions in solution.1 Covalent extrinsic
fluorophores such as dansyl, or intrinsic fluorophores such as
tryptophan, are commonly used for this purpose, each with
serious limitations, however. The use of extrinsic probes
requires post-translational modification, which is often nonspe-
cific and/or inefficient, although reactive lysine or cysteine
residues can sometimes be introduced or removed, as needed,
by site-directed mutagenesis.2 The use of intrinsic tryptophan
residues is problematic when multiple tryptophans are present,
although the fluorescence spectra can be simplified by the
conservative replacement of all but one tryptophan residue with
phenylalanine or tyrosine using site-directed mutagenesis.3 This
strategy always carries the risk, however, that changing multiple
residues will have unanticipated effects on protein structure and/
or function.4
The introduction of noncoded amino acids into proteins by
in Vitro suppression of termination codons5 offers a unique
* Authors to whom correspondence should be addressed.
† University of California, Irvine.
‡ Mt. Sinai School of Medicine.
analogues have been incorporated into proteins by in ViVo
methods that uniformly replace all of the tryptophan residues.6
Dansyl has been introduced post-translationally as a sulfonyl
halide or isothiocyanate, but it generally modifies nucleophilic
amino groups indiscriminately.7,8 We chose to incorporate these
amino acids into â-galactosidase because it is a difficult case
with multiple tryptophan and lysine residues (37 and 20,
respectively, per subunit)9 and because incorporation of non-
coded residues is readily monitored, as described below.
Suppressor tRNA aminoacylated with 7-azaTrp was prepared
by our standard methodology.5a,b Boc-7-azaTrp was prepared
X Abstract published in AdVance ACS Abstracts, December 15, 1996.
(1) (a) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum
Press: New York, 1983. (b) Topics in Fluorescence Spectroscopy;
Lakowicz, J. R., Ed.; Plenum Press: New York, 1991-1992; Vol. 1-3.
(c) Beechem, J. M.; Brand, L. Ann. ReV. Biochem. 1985, 54, 43-71.
(2) Mannuzzu, L. M.; Moronne, M. M.; Isacoff, E. Y. Science 1996,
271, 213-216.
(3) (a) Royer, C. A.; Gardner, J. A.; Beechem, J. M.; Brochon, J. C.;
Matthews, K. S. Biophys. J. 1990, 58, 363-378. (b) Harris, D. L.; Hudson,
B. S. Biochemistry 1990, 29, 5276-5285. (c) Hasselbacher, C. A.; Rusinova,
R.; Rusinova, E.; Ross, J. B. A. In Techniques in Protein Chemistry VI;
Crabb, J. W., Ed.; Academic Press: New York, 1995; pp 349-356.
(4) Difference spectroscopy is one powerful technique that has recently
been developed for deciphering intrinsic Trp fluorescence in multi-Trp
proteins. See: Hasselbacher, C. A.; Rusinova, E.; Waxman, E.; Rusinova,
R.; Kohanski, R. A.; Lam, W.; Guha, A.; Du, J.; Lin, T. C.; Polikarpov, I.;
Boys, C. W. G.; Nemerson, Y.; Konigsberg, W. H.; Ross, J. B. A. Biophys.
J. 1995, 69, 20-29.
(5) (a) Steward, L. E.; Chamberlin, A. R. In Methods in Molecular
Biology. Protein Synthesis: Methods and Protocols; Martin, R., Ed.;
Humana Press: New Jersey, in Press. (b) Steward, L. E.; Chamberlin, A.
R. In Encyclopedia of Molecular Biology and Molecular Medicine; Meyers,
R. A., Ed.; VCH Publishers: New York, in press. (c) Mendel, D.; Cornish,
V. W.; Schultz, P. G. Annu. ReV. Biophys. Biomol. Struct. 1995, 24, 435-
462.
(6) (a) This has been accomplished with Trp deficient bacterial auxo-
trophs. See Ross, J. B. A.; Szabo, A. G.; Hogue, C. W. V. Methods Enzymol.,
in press, and references cited therein. (b) The site-specific incorporation of
amino acid 3 into T4 lysozyme by suppression techniques has also
previously been reported: Cornish, V. W.; Benson, D. R.; Altenbach, C.
A.; Hideg, K.; Hubbell, W. L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 2910-2914.
(7) Examination of the X-ray crystal structure reveals that the majority
of the 20 Lys residues per monomer are on the protein surface: Jacobson,
R. H.; Zhang, X.-J.; DuBose, R. F.; Matthews, B. W. Nature 1994, 369,
761-766.
S0002-7863(96)03023-5 CCC: $14.00 © 1997 American Chemical Society

Fluorescent Spectroscopy of â-Galactosidase
J. Am. Chem. Soc., Vol. 119, No. 1, 1997 7
essentially as described by Rich et al.10 and then coupled with
pdCpA (CDI) to give Boc-7-azaTrp-pdCpA. The tert-butyl
group was removed by treatment with 90:5:3:2 TFA/thioanisole/
ethanedithiol/anisole for 10 min at ambient temperature, fol-
with the noncoded amino acid of interest yields full-length
mutant â-galactosidase modified at position 7. Since mutations
in this region are known not to affect enzymatic activity,13
production of â-galactosidase is monitored very conveniently
by the well-established colorimetric assay for enzymatic
activity.5a,14
lowed by ligation of the fully deprotected aminoacyl dinucleo-
Gly
tide to tRNA -COH to give the desired suppressor, 7-azaTrp-
CUA
tRNAGCUlyA-dCA. The ꢀ-dnsLys suppressor was prepared in a
similar fashion with RN-Boc-ꢀN-dansylLys in place of the
tryptophan derivative; full details are given in the Experimental
Low protein yield, compared to in ViVo expression methods,
is a major drawback to in Vitro protein synthesis. The yields
of even wild-type proteins are relatively low in S30 lysates,
and suppression mutagenesis lowers the yields even more. The
reduced levels of full-length protein are in part a result of the
competition for binding to the termination codon between the
suppressor tRNAs and the endogenous release factors (RFs) that
normally mediate termination of translation. We demonstrated
previously that when noncoded amino acids are incorporated
by expansion of the genetic code, i.e., using a non-natural codon
that is not recognized by RFs, the level of suppression is
increased.15 While this method of expanding the genetic code
is not amenable to the synthesis of large proteins, the results
did suggest that we might be able to garner the same benefits
by deactivating or removing a release factor present in the S30
lysate. We therefore prepared lysate from a mutant strain
(US477)16 of E. coli that produces a faulty RF1 (which is the
release factor competing with our suppressors for binding to
the AUG codon), hypothesizing that heat shock treatment of
this lysate prior to the translation reaction might thermally
denature RF1 and lead to increased suppression efficiencies.
Section.
Gly
CUA
Preparation of the 5-OHTrp-tRNA -dCA suppressor
proved to be considerably more difficult. Initially, amino-
acylation of the dinucleotide was attempted by the standard CDI
acylation method; however, the major product recovered from
this reaction was the acyl imidazole derivative resulting from
competitive reaction of the (unprotected) 5-OH group. Addi-
tives such as pentachlorophenol that reportedly suppress esteri-
fication of the indole hydroxyl group in solid-phase peptide
synthesis11 failed to resolve the problem in this case. Acylation
reactions with the indole hydroxyl group protected as the
tetrabutyldimethyl silyl ether (TBS) gave no desired product,
while in situ protection of the hydroxyl as the trimethylsilyl
(TMS) ether followed by CDI activation and coupling to pdCpA
gave the desired product in only 1% yield.
We finally resorted to blocking the R-amine with the
photolabile Nvoc protecting group, which allows the acylation
to be conducted in dry organic solvent and avoids the necessity
of treating the sensitive tryptophan analogue with TFA in the
nitrogen deprotection step. The Nvoc-5-OHTrp was prepared
from 5-OHTrp essentially as described for other amino acids,12
followed by triethylsilylation of the 5-hydroxy group. Activa-
tion of the carboxyl group as the cyanomethyl ester, coupling
with the tetrabutylammonium salt of the dinucleotide in dry
DMF, and in situ desilylation with aqueous acetic acid gave
the 2′- and 3′-isomers in 15% yield after reverse-phase HPLC
Preliminary experiments showed that, as predicted, heat-
shocked S30 extracts prepared from this strain lead to increased
amounts of suppression product compared to extracts from
standard Escherichia coli strains,17 so that subsequent suppres-
sion reactions were run with US477 S30 lysates containing the
DNA template pT7lac-7amb and amber suppressor tRNA
charged with amino acids 1-4. Upon completion, the crude
reactions were checked for â-galactosidase activity via the
standard o-(nitrophenyl)-â-D-galactopyranoside hydrolysis
assay.5a,14 The control amino acid, phenylalanine (1), and all
three noncoded amino acids were found to be incorporated well
above background levels (Table 1). Control reactions containing
(i) no template and no suppressor tRNA, (ii) template but no
suppressor tRNA, and (iii) template with non-acylated suppres-
sor tRNA produced little or no active â-galactosidase. There-
fore, any â-galactosidase produced above background levels
within the suppression reactions is due to suppression of the
termination codon by aminoacyl suppressor tRNA, resulting in
incorporation of the charged amino acid into â-galactosidase.
On the basis of previous incorporation studies with both E. coli
auxotrophs6a and suppressors,6b it was not surprising that
analogues 2 and 3 were incorporated efficiently. We were
gratified to find that 4 was also incorporated well above
purification. This aminoacyl dinucleotide was then ligated to
Gly
tRNA -COH, and the Nvoc group was photolytically re-
CUA
moved as described in the Experimental Section to yield the
desired suppressor, 5-OHTrp-tRNAGCUlyA-dCA.
With the three synthetic suppressors in hand, we turned to
determining their suppression efficiencies in protein synthesis.
As a test case we chose â-galactosidase, which we routinely
use as an initial screen for the biosynthetic incorporation of
noncoded amino acids by suppression of amber stop codons.
The plasmid construct pT7lac-7amb, which contains a DNA
sequence that corresponds to the lacZ gene with an inserted
TAG (amber) stop codon at position 7, serves as the DNA
template for incorporation of noncoded amino acids into
â-galactosidase. Transcription and translation of the DNA
template in the presence of synthetic suppressor tRNA5a charged
(8) It should be mentioned that other amino acids modified with
fluorescent reagents have also been incorporated biosynthetically, but either
in systems that do not allow site-specificity or in systems that are more
limited. See: (a) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.;
Johnson, A. E. Cell 1994, 78, 461-471. (b) Crowley, K. S.; Reinhart, G.
D.; Johnson, A. E. Cell 1993, 73, 1101-1115. (c) Picking, W. D.; Picking,
W. L.; Odom, O. W.; Hardesty, B. Biochemistry 1992, 31, 2368-2375.
(d) Picking, W. D.; Odom, O. W.; Hardesty, B. Biochemistry 1992, 31,
12565-12570 and references cited therein.
(13) Studies of lacZ gene fusions have shown that up to 26 amino acids
can be removed from the amino terminus and can be substituted by other
amino acid sequences with little or no effect on the specific activity: Fowler,
A. V.; Zabin, I. J. Biol. Chem. 1983, 258, 14354-14358. This allows
noncoded amino acids to be screened efficiently without fear that decreased
activity is due to structural changes. Additionally, since termination competes
with suppression to give only a hexapeptide, there is no possibility of the
termination product having enzymatic activity.
(14) Miller, J. H. Experiments in Molecular Genetics; Cold Spring Harbor
Laboratory: Cold Spring Harbor, New York; 1972, pp 352-359, 403-
404.
(15) Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A. Nature
1992, 356, 537-539.
(9) The active enzyme is a homotetramer comprised of 116 kDa (1023
amino acid) monomers. See: (a) Fowler, A. V.; Zabin, I. J. Biol. Chem.
1978, 253, 5521-5525. (b) Kalnins, A.; Otto, R.; Ruther, U.; Muller-Hill,
B. Embo J. 1983, 2, 593-597.
(10) Rich, R. L.; Smirnov, A. V.; Schwabacher, A. W.; Petrich, J. W. J.
Am. Chem. Soc. 1995, 117, 11850-11853.
(16) (a) Ryde´n, S. M.; Isaksson, L. A. Mol. Gen. Genet. 1984, 193, 38-
45. (b) Ryde´n, M.; Murphy, J.; Martin, R.; Isaksson, L.; Gallant, J. J.
Bacteriol. 1986, 168, 1066-1069. (c) Zhang, S.; Ryde´n-Aulin, M.;
Kirsebom, L. A.; Isaksson, L. A. J. Mol. Biol. 1994, 242, 614-618.
(17) A detailed comparision is underway.
(11) Martinez, J.; Bodanszky, M. Int. J. Peptide Protein Res. 1978, 12,
277-283.
(12) Ellman, J.; Mendel, D.; Anthony-Cahill, S.; Noren, C. J.; Schultz,
P. G. Methods Enzymol. 1991, 202, 301-336.

8
J. Am. Chem. Soc., Vol. 119, No. 1, 1997
Steward et al.
Table 1. Yields for 50 µL Lysate Reactions
DNA templatea
suppressor
µg of â-galb
pT7lac (wild type)
pT7lac-7amb
pT7lac-7amb
pT7lac-7amb
pT7lac-7amb
pT7lac-7amb
2.71 ( 0.21
Phe-tRNAGCUlyA-dCA
0.780 ( 0.075
5-OHTrp-tRNAGCUlyA-dCA
7-azaTrp-tRNAGCUlyA-dCA
ꢀ-dnsLys-tRNAGCUlyA-dCA
0.353 ( 0.015
0.227 ( 0.006
0.089 ( 0.014
0.014 ( 0.003
tRNAGCUlyA-dCAc
pT7lac-7amb
e
d
0.003 ( 0.001
0.001 ( 0.001
a DNA templates were either the plasmid pT7lac containing the lacZ
gene for full-length wild-type â-galactosidase or the plasmid pT7lac-
7amb containing the lacZ gene with an amber stop codon at position
7. b Yields are determined as described in the text and are averages for
three assays of crude lysate reaction. c Non-acylated suppressor tRNA.
d Control reaction without suppressor tRNA. e Control reaction lacking
plasmid and suppressor tRNA.
Figure 1. Fluorescence spectra (uncorrected) of dnsLys-â-galactosidase
(13.6 nM, s), Phe-â-galactosidase (21.4 nM, - -), and wild-type
â-galactosidase purified from ꢀ-dnsLys (10.5 nM, - - -); excitation at
340 nm.
background levels, albeit with lower efficiency than 1-3, despite
literature reports to the contrary for a related translation
system.6b,18
Finally, since the experimental estimates of concentration by
fluorescence and by enzymatic activity agree within 20%, it
seems reasonable to conclude that the amount of quenching by
a single dansyl, or the incremental increase in fluorescence due
to a single 5-OHTrp, would affect the total protein emission by
only a few percent. There would, of course, be much greater
uncertainty in this regard if the protein had only three or four
tryptophan residues, which is another advantage of employing
a tryptophan-rich enzyme such as â-galactosidase for these initial
studies.
We demonstrated previously that red-edge excitation at 315
nm could be used to selectively excite the fluorescence of
5-OHTrp in the protein λ cI repressor, which has only three
tryptophan residues.20 In the case of 5-OHTrp-â-galactosidase,
even with this mode of excitation, the fluorescence yield of the
incorporated tryptophan analogue was too low compared to that
of the 37 tryptophan residues to clearly resolve the analogue
emission. On the other hand, the dansyl group has a lower
energy absorption band that allows excitation at 340 nm, and
we were able to obtain a well-resolved emission spectrum from
dnsLys-â-galactosidase (Figure 1). The spectrum shows a
fluorescence emission maximum at ca. 500 nm, which is shifted
from that of ꢀ-dnsLys in water21 and corresponds well with
published emission maxima for proteins that have been non-
specifically modified post-translationally with dansyl chloride.22
As anticipated, the fluorescence spectrum of the control mutant
Phe-â-galactosidase reveals no fluorescence in the emission
region of a dansyl group. The fluorescence spectrum of dnsLys-
â-galactosidase in conjunction with the enzymatic activity
confirms the incorporation of ꢀ-dnsLys into a full-length,
functional protein.
Amounts of 5-OHTrp- and ꢀ-dnsLys-â-galactosidase suf-
ficient for fluorescence analysis were purified from in Vitro
suppression reactions by affinity chromatography.19 The mutant
proteins were assayed for activity and their approximate
concentrations determined.5a,14 Relative concentrations were
determined by fluorescence, based on comparing the dominant
tryptophan emission after excitation at 280 and 295 nm (the
amounts of protein were too small to quantify accurately by
absorption). As controls, wild-type â-galactosidase was purified
from lysate to which each fluorescent amino acid analogue had
been added post-reaction, and Phe-â-galactosidase was prepared
from the corresponding phenylalanyl suppressor, purified, and
characterized in parallel with the other mutant proteins.
It should be noted that dansyl and 5-OHTrp also are excited
at 280 and 295 nm, which could affect this quantitation method.
Dansyl fluorescence would not interfere directly because it is
well-separated from that of tryptophan; however, the spectral
overlap of the dansyl absorption with tryptophan fluorescence
does allow the possibility of resonance energy transfer, which
would cause quenching of the tryptophan emission. In the case
of 5-OHTrp, its fluorescence and that of tryptophan do occur
at similar wavelengths, and therefore, the analogue will con-
tribute to the total emission. While it is difficult to predict a
priori the fluorescence yield of any fluorophore in a protein
environment, it is useful to consider what might be expected
for two different fluorophores given similar environments. For
example, in water at pH 7, the free amino acids tryptophan and
5-OHTrp have average fluorescence lifetimes of approximately
3 and 3.7 ns.6a,20 With these considerations in mind, it should
be recalled that â-galactosidase has 37 tryptophan residues per
subunit; assuming to a first-order approximation that the
fluorescence yields for a tryptophan and a 5-OHTrp residue in
the protein are equivalent, the analogue would be expected to
make only a ca. 3% contribution to the total emission.
Conversely, dansyl will tend to quench the nearest tryptophan
residues, and while the magnitude of dansyl quenching is
impossible to predict accurately, the net result would most likely
be a less than 5% reduction in the emission, considering the
size of the protein and the number of typtophan residues.
The results described in this paper clearly illustrate the ability
to site-specifically engineer spectrally enhanced proteins with
minimal structural perturbations. We are particularly interested
in developing in Vitro, site-specific incorporation of fluorescence
probes to investigate the functional interactions of proteins
(21) Haugland, R. P. Molecular Probes Handbook of Fluorescent Probes
and Research Chemicals, 5th ed.; Molecular Probes, Inc: Eugene, OR, 1992.
(22) (a) Vehar, G. A.; Reddy, A. V.; Freisheim, J. H. Biochemistry 1976,
15, 2512-2518. (b) Grossman, S. H. Biochim. Biophys. Acta 1984, 785,
61-67. (c) Kincaid, R. L.; Billingsley, M. L.; Vaughan, M. Methods
Enzymol. 1988, 159, 605-626. (d) As an additional control for hydrophobic
binding (or adsorption) of ꢀ-dnsLys, wild-type â-galactosidase produced
in a control S-30 lysate reaction was incubated in the presence of ꢀ-dnsLys.
The wt enzyme was then purified as described and the fluorescence spectrum
recorded. It is shown, in figure 1, that no significant amount of ꢀ-dnsLys
is present in the purified wt â-galactosidase, further evidence that the
fluorescence emission of dnsLys-â-galactosidase is due to ꢀ-dnsLys that is
incorporated into the peptide chain.
(18) This difference could be due to different proteins or different codon
context, as well as the use of the temperature-sensitive RF strain.
(19) Steers, E., Jr.; Cuatrecasas, P. Methods Enzymol. 1974, 34, 350-
358.
(20) Ross, J. B. A.; Senear, D. F.; Waxman, E.; Kombo, B. B.; Rusinova,
E.; Huang, Y. T.; Laws, W. R.; Hasselbacher, C. A. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 12023-12027.

Fluorescent Spectroscopy of â-Galactosidase
J. Am. Chem. Soc., Vol. 119, No. 1, 1997 9
of pdCpA dissolved in 0.25 mL of 100 mM NaH2PO4 was added,
resulting in formation of a white precipitate that dissolved after ca. 10
min of vigorous strirring. The reaction was stirred for 1 h and then
was extracted with 0.75 mL of EtOAc. The organics were back-
extracted with 10 mM AcOH (2 × 0.2 mL). The combined aqueous
extracts were acidified with glacial AcOH (pH e 5), yielding a small
amount of precipitate that was dissolved by the addition of CH3CN to
10% (v/v). The sample was filtered and purified by semipreparative
reverse phase HPLC using a gradient of 5:95 f 60:40 CH3CN/10 mM
AcOH over 55 min [retention time (rt) ) 28.6 and 29.7 min]25 to afford
0.002 mmol (11% yield) of 1a. The purified product was lyophilized
and stored at -75 °C. High-resolution MS calcd for C33H44N9O16P2
involved in blood coagulation and regulation of transcription;
however, we anticipate that this approach would be general for
fluorescence spectroscopy of any protein that can be expressed
successfully in Vitro.
Experimental Section
General Methods. Unless otherwise noted, reagents were obtained
from commercial suppliers and were used without further purification.
L-5-Hydroxytryptophan, D,L-7-azatryptophan, ꢀN-dansyl-L-lysine, Dowex
50W-X8-200 ion exchange resin, and the â-galactosidase affinity resin
p-aminobenzyl 1-thio-â-D-galactopyranoside supported on 4% beaded
agarose were purchased from Sigma, Boc-L-phenylalanine was from
BaChem, and tetrabutylammonium hydroxide, [40% (w/w)] was from
Aldrich. Acetonitrile (CH3CN) and dimethylformamide (DMF) were
dried over activated 4 Å molecular sieves. Triethylamine (Et3N) was
purified by distillation from calcium hydride. Trifluoroacetic acid
(TFA) was dried by distillation from P2O5. All moisture-sensitive
reactions were performed under positive pressure of nitrogen in flame-
or oven-dried glassware. All aqueous solutions were prepared from
water filtered through a Millipore Nanopure purification system. 1H-
NMR spectra were obtained on an Omega 500 (500 MHz) or a General
Electric QE-300 (300 MHz) spectrometer. Spectral data are reported
in ppm relative to the solvent peak. Data are reported as follows:
chemical shift, multiplicity (app ) apparent, par obsc ) partially
obscured, ovrlp ) overlapping, s ) singlet, d ) doublet, t ) triplet, q
) quartet, dd ) doublet of doublets, m ) multiplet, br ) broad),
coupling constant(s) in Hz, and integration. Infrared (IR) spectra were
recorded as thin films with a Perkin-Elmer Model 1600 series FTIR
spectrophotometer. Fast atom bombardment mass spectra (FAB-MS)
were recorded at the U.C. Irvine Mass Spectral Laboratory on a Fisions
VG AutoSpec. Absorbance spectra were recorded on a Kontron
Instruments UVIKON 9410 UV-vis spectrophotometer. Fluorescence
spectra were recorded on Hitachi F-4500 and SLM-4800 fluorescence
spectrophotometers. Thin-layer chromatography (TLC) was performed
on 0.25 mm Merck precoated silica gel plates (60 F-254), and flash
chromatography was performed using ICN 200-400 mesh silica gel.
Reverse-phase high-performance liquid chromatography (rpHPLC) was
performed on a Rainin Dynamax system consisting of two model SD-
200 pumps and an in-line Rainin Dynamax UV-1 absorbance detector
interfaced with a Macintosh LC II. Analytical HPLC was performed
with a Microsorb-MV C-18 analytical column (5 µm packing, 4.6 mm
i.d. × 250 mm length) at a flow rate of 1.0 mL/min, and semiprepartive
HPLC was perfomed with a Waters Nova-Pak C-18 semipreparative
column (5 µm packing, 10 mm i.d. × 250 mm length) at a flow rate
of 5 mL/min.
(M + H+) 884.2381, found 884.2402 (FAB, 3:1 glycerol/thioglycerol).
Gly
L-Phenylalanyl-tRNA -dCA (1b). Deprotection of aminoacyl
CUA
Gly
CUA
dinucleotide 1a and ligation to 74-mer tRNA -COH was carried out
as previously reported, with the following modifications.5a,b Im-
mediately following termination of the ligation reaction with 3 M
NaOAc, 40 µg of glycogen was added and 1b was precipitated by the
addition of 3 vol of absolute EtOH and incubation at -75 °C for 90
min. The precipitate was pelleted by centrifugation at 14000g for 30
min at 4 °C. The pellet was washed (2×) with 70:30 EtOH/10 mM
AcOH and dried in Vacuo with a SpeedVac system. The dry pellet
was resuspended in 150 µL of 10 mM AcOH, and insoluble material
was removed by centrifugation. The cleared supernatant was quantified
by UV absorbance, lyophilized to dryness, and stored at -75 °C until
use.
N-(Nvoc)-L-5-Hydroxytryptophan (2a). To a stirred solution of
750 mg (2.93 mmol) of L-5-hydroxytryptophan‚dihydrate in 15 mL of
a 2:1 mixture of 0.3 M aqueous Na2CO3/dioxane at ambient temperature
was added dropwise [(6-nitroveratryl)oxy]carbonyl chloride26 as a
suspension in dioxane (7 mL). The reaction was stirred for 2 h. The
reaction was made basic by addition of 20 mL of saturated Na2CO3
and was extracted with CH2Cl2 (3 × 20 mL). The aqueous layer was
acidified to pH 2 with 1 M HCl and extracted with EtOAc (3 × 30
mL). The combined organic extracts were dried over MgSO4, filtered,
and concentrated in Vacuo to yield a yellow oil. The oil was
resuspended in EtOAc, adsorbed onto SiO2, and purified by flash
chromatography on SiO2 (1.8:98:0.2 MeOH/CH2Cl2/AcOH f 5.8:94:
0.2 MeOH/CH2Cl2/AcOH) to give 863.1 mg (67.6% yield) of 2a as a
yellow oil: 1H-NMR (300 MHz, CD3OD) δ 7.68 (s, 1H) 7.14 (d, J )
8.6 Hz, 1H), 7.05 (s, 1H), 7.00 (s, 1H), 6.95 (d, J ) 2.0, 1H), 6.64 (dd,
J ) 2.0, 8.6 Hz, 1H), 5.40 (d, J ) 2.5 Hz, 2H), 4.48 (dd, J ) 8.7, 4.5
Hz, 1H), 3.86 (s, 3H), 3.73 (s, 3H), 3.29 (ovrlp m, 1H), 3.07 (dd, J )
8.7, 14.9 Hz, 1H); IR (thin film) 3385, 2933, 2361, 1700, 1582, 1522,
1456, 1439, 1326, 1278, 1220, 1071 cm-1; high-resolution MS calcd
for C21H21N3O9 (M+) 459.1278, found 459.1271 (FAB, m-nitrobenzyl
alcohol).
N-(Nvoc)-5-[(Triethylsilyl)oxy]-L-tryptophan (2b). To a stirred
solution of 455 mg (0.99 mmol) of 2a and 0.61 mL (5.27 mmol) of
lutidine in 10 mL of anhydrous CH3CN at 0 °C was added dropwise
0.60 mL (2.64 mmol) of triethylsilyl triflate. After 2 h, the reaction
was diluted with 30 mL of EtOAc, extracted with H2O (2 × 10 mL),
and washed with brine (10 mL). The organics were dried over MgSO4,
filtered, and concentrated in Vacuo to yield a yellow oil. Purification
by flash chromatography on SiO2 (2:98 MeOH/CH2Cl2 f 8:92 MeOH/
CH2Cl2) gave 274 mg (48% yield) of 2b as a yellow oil: 1H-NMR
(300 MHz, CD3OD) δ 8.19 (br s, 1H), 7.69 (s, 1H), 7.19 (d, J ) 8.7
Hz, 1H), 7.02 (d, J ) 1.4 Hz, 1H), 6.98 (s, 1H), 6.89 (s, 1H), 6.77 (dd,
J ) 1.4, 8.7 Hz, 1H), 5.60 (d, J ) 15.3 Hz, 1H), 5.44 (d, J ) 15.3 Hz,
1H), 5.39 (par obsc m, 1H), 4.74 (m, 1H), 3.93 (s, 3H), 3.82 (s, 3H),
3.31 (d, J ) 5.1 Hz, 2H), 0.98 (t, J ) 7.8 Hz, 9H), 0.72 (q, J ) 7.8
Hz, 6 H); IR (thin film) 3388, 2956, 2881, 2360, 1714, 1581, 1520,
1476, 1328, 1278, 1220, 1073, 956 cm-1; high-resolution MS calcd
for C27H35N3O9Si (M+) 573.2142, found 573.2151 (FAB, m-nitrobenzyl
alcohol).
The dinucleotide, 5′-O-phosphoryl-2′-deoxycytidylyl-(3′-5′)-adeno-
sine (pdCpA), was synthesized as described,5a,b and the truncated 74-
Gly
CUA
mer tRNA (tRNA -COH) was prepared by runoff transcription of
plasmid pJDB2, as reported previously.5a,b Photodeprotections were
accomplished with a Pyrex-jacketed Hanovia 300 W mercury lamp.
Preparation of the Tetrabutylammonium Salt of pdCpA. The
tetrabutylammonium salt of the dinucleotide was prepared with 50W-
X8-200 ion exchange resin in the tetrabutylammonium form.
A
column containing ca. 5 mL swelled volume of resin was equilibrated
with 1% tetrabutylammonium hydroxide until the eluant was basic (pH
12-13), and the column was then washed extensively with H2O until
the pH of the eluant was neutral. Dinucleotide pdCpA (100 mg) was
dissolved in 0.5 mL of H2O, loaded onto the column, and eluted with
H2O. Fractions (1-2 mL) were collected, and elution was followed
by UV absorbance of material spotted on TLC plates. After elution,
0.2 equiv of tetrabutylammonium hydroxide (40% w/w) was added to
the dinucleotide.23 The sample was then lyophilized to dryness and
stored at -20 °C.
5′-O-Phosphoryl-2′-deoxycytidylyl-(3′-5′)-2′(3′)-O-[N-(Boc)-L-
phenylalanyl]adenosine (1a). Aminoacylation was carried out ac-
cording to the method of Gottikh,24 with minor modifications. To a
stirred solution of 45.7 mg (0.17 mmol) of Boc-L-phenylalanine in 0.25
mL of dry CH3CN at ambient temperature was added 37.4 mg (0.23
mmol) of carbonyl diimidazole. After 20 min, 11.1 mg (0.017 mmol)
(24) See, for example: (a) Gottikh, B. P.; Krayevsky, A.; Tarussova, N.
B.; Purygin, P. P.; Tsilevich, T. L. Tetrahedron 1970, 26, 4419-4433. (b)
Tarusova, N. B.; Mazurova, V. V.; Kraevskii, A. A.; Gottikh, B. P. Bull.
Acad. Sci. USSR, DiV. Chem. Sci. 1971, 1630-1633 and preceeding paper
in the series.
(25) The two peaks correspond to the 2′- and 3′- isomers.
(26) Amit, B.; Sehavi, V.; Patchornik, A. J. Org. Chem. 1974, 39, 192-
196.
(23) Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc.
1991, 113, 2722-2729.

10 J. Am. Chem. Soc., Vol. 119, No. 1, 1997
Steward et al.
N-(Nvoc)-5-[(triethylsilyl)oxy]-L-tryptophan Cyanomethyl Ester
(2c). To a solution of 190 mg (0.331 mmol) of amino acid 2b in 0.3
mL of dry DMF at ambient temperature were added 0.38 mL (2.75
mmol) of triethylamine and 0.255 mL (4.0 mmol) of chloroacetonitrile.
The reaction was stirred overnight. The reaction was diluted with 25
mL of CH2Cl2, extracted with H2O (2 × 25 mL), and washed with
brine (25 mL). The organics were dried over MgSO4, filtered, and
concentrated in Vacuo to a yellow oil. The oil was diluted with CH2-
Cl2, adsorbed onto SiO2, and purified by flash chromatography on SiO2
(1:2 EtOAc/hexanes f 1:1 EtOAc/hexanes) to give 167.4 mg (82.6%
yield) of 2c: 1H-NMR (300 MHz, CDCl3) δ 8.06 (br s, 1H), 7.70 (s,
1H), 7.22 (d, J ) 8.7 Hz, 1H), 7.03 (d, J ) 2.2 Hz, 1H), 6.98 (s, 1H),
6.90 (s, 1H), 6.79 (dd, J ) 2.2, 8.7 Hz, 1H), 5.61 (d, J ) 15.4 Hz,
1H), 5.42 (ovrlp d, J ) 15.4 Hz, 1H), 5.37 (d, J ) 8.9 Hz, 1H), 4.79
(par obsc m, 1H), 4.79 (d, J ) 15.6 Hz, 1H), 4.65 (d, J ) 15.6 Hz,
1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.31 (d, J ) 5.4 Hz, 2H), 0.99 (t, J )
7.8 Hz, 9H), 0.73 (q, J ) 7.8 Hz, 6H); IR (thin film) 3388, 2956,
added, resulting in a thick yellow paste. The reaction was stirred for
1 h and then was extracted with 1 mL of EtOAc. The organics were
back-extracted with 10 mM AcOH (2 × 0.2 mL). The combined
aqueous extracts were acidified with glacial AcOH (pH e 5), yielding
a precipitate that was dissolved by the addition of CH3CN to 10% (v/
v). The sample was filtered and purified by semipreparative reverse-
phase HPLC using a gradient of 10:90 f 60:40 CH3CN/10 mM AcOH
over 50 min (rt ) 22.5 and 23.1 min) to afford 0.74 µmol (2.3% yield)
of 3b. The purified product was lyophilized and stored at -75 °C.
High-resolution MS calcd for C34H43N11O16P2Na (M + Na+) 946.2262,
found 946.2253 (FAB 3:1 glycerol/thioglycerol).
Gly
CUA
D,L-7-Azatryptophanyl-tRNA -dCA (3c). Aminoacyl dinucle-
otide 3b was Boc-deprotected immediately prior to ligation as
described,5a with the following modifications. Lyophilized 3b was
resuspended in 200 µL of reagent R (90:5:3:2 TFA/thioanisole/
ethanedithiol/anisole) and incubated at ambient temperature for 10 min.
The deprotected material was precipitated by addition of 2 mL of Et2O
and pelleted by centrifugation (14000g). The pellet was resuspended
in 10 mM AcOH (100 µL), concentrated to dryness in Vacuo on a
1760, 1715, 1581, 1520, 1476, 1329, 1278, 1220, 1170, 1073 cm-1
;
high-resolution MS calcd for C29H37N4O9Si (M+) 612.2251, found
612.2247 (FAB, m-nitrobenzyl alcohol).
SpeedVac system, and resuspended and concentrated a second time.
Gly
5′-O-Phosphoryl-2′-deoxycytidylyl-(3′-5′)-2′(3′)-O-[N-(Nvoc)-L-5-
hydroxytryptophanyl]adenosine (2d). A 1 dram vial containing a
stir bar and 51 mg (0.084 mmol) of the activated ester 2c and 21.2 mg
(0.018 mmol) of the tetrabutylammonium salt of pdCpA was dried
overnight in a vacuum desiccator containing P2O5. To the dried reagents
was added 0.5 mL of dry DMF, and the reaction was stirred at ambient
temperature for 3.5 h. The reaction was quenched with 1 mL of 10 M
aqueous AcOH, yielding a precipitate that was solubilized by addition
of 0.3 mL of CH3CN. The reaction was stirred at ambient temperature
for 40 min, and then 50 mM NH4OAc (pH 5.0) was added until a small
amount of precipitate formed. The precipitate was removed by
centrifugation in a microcentrifuge tube. The cleared supernatant was
filtered and partially purified in four semipreparative reverse-phase
HPLC runs with a gradient of 25:75 f 80:20 CH3CN/50 mM NH4-
OAc (pH 5.0) over 55 min. Products eluting between 6 and 14 min
were combined and lyophilized. The partially purified material was
resuspended in 20:80 CH3CN/10 mM AcOH. The sample was desalted
and purified by semipreparative HPLC with a gradient of 15:85 f 60:
40 CH3CN/10 mM AcOH (rt ) 16, 19 and 21 min). FAB-MS analysis
revealed that product eluting at 16 min contained tetrabutylammonium
ion. This material was lyophilized and desalted by reverse-phase HPLC
with the same CH3CN/10 mM AcOH gradient. The purified products
were combined and lyophilized to give 2.9 mg (15.2% yield) of 2d.
Product was stored at -75 °C as a lyophilized powder. MS calcd for
C40H46N11O21P2 (M + H+) 1078, found 1078 (FAB, 3:1 glycerol/
The deprotected material was then ligated to 74-mer tRNA -COH as
CUA
described.5a
rN-(Boc)-EN-(dansyl)-L-lysine (4a). To a stirred solution of 150
mg (0.39 mmol) of 4 and 330 mg (2.39 mmol) of K2CO3 in 1.5 mL of
1:2 H2O/dioxane cooled to 0 °C was added di-tert-butyldicarbonate in
0.5 mL of dioxane. After 1 h the reaction was allowed to warm to
ambient temperature and was stirred overnight. The reaction was
acidified to pH 4.0 with saturated citric acid and was extracted with
EtOAc (3 × 5 mL). The combined organics were washed with brine,
dried over MgSO4, filtered, and concentrated in Vacuo. The fluorescent
oil was dissolved in EtOAc, adsorbed onto SiO2, and purified by flash
chromatography on SiO2 (29:70:1 EtOAc/hexanes/AcOH f 59:40:1
EtOAc/hexanes/AcOH) to yield 181 mg (97% yield) of 4a as a
fluorescent yellowish-green oil: 1H-NMR (500 MHz, CDCl3) δ 8.52
(d, J ) 8.4 Hz, 1H), 8.29 (d, J ) 8.4 Hz, 1H), 8.22 (d, J ) 7.2 Hz,
1H), 7.55-7.49 (m, 2H), 7.18 (d, J ) 7.6 Hz, 1H), 5.22 (s, 1H), 5.13
(d, J ) 7.6 Hz, 1H), 4.20 (d, J ) 5.2 Hz, 1H), 2.88 (app s, 8H), 1.69-
1.66 (br, 2H), 1.55-1.53 (br, 2H), 1.43 (s, 9H), 1.35-1.28 (br, 2H);
IR (thin film) 3287, 2939, 2871, 2354, 1712, 1694, 1568, 1506, 1452,
1392, 1367, 1315, 1232, 1160, 1145, 1073 cm-1; high-resolution MS
calcd for C23H33N3O6S (M+) 479.2090, found 479.2080 (FAB, m-
nitrobenzyl alcohol).
5′-O-Phosphoryl-2′-deoxycytidylyl-(3′-5′)-2′(3′)-O-[RN-(Boc)-ꢀN-
(dansyl)-L-lysinyl]adenosine (4b). To a stirred solution of 29.1 mg
(0.06 mmol) of 4a in 0.1 mL of dry CH3CN at ambient temperature
was added 12.9 mg (0.08 mmol) of carbonyldiimidazole. After 20 min
3.9 mg (0.006 mmol) of pdCpA in 0.5 mL of 100 mM NaH2PO4 was
added, causing a yellow precipitate to form. The precipitate dissolved
after 10 min of vigorous stirring. The reaction was stirred an additional
50 min and then was extracted with 1 mL of EtOAc. The organics
were back-extracted with 10 mM AcOH (2 × 0.2 mL). The combined
aqueous extracts were acidified with glacial AcOH (pH e 5), yielding
a precipitate that was dissolved by addition of CH3CN to 10% (v/v).
The sample was filtered and purified by semipreparative reverse-phase
HPLC with a gradient of 16:84 to 60:40 CH3CN/10 mM AcOH over
44 min (rt ) 24.5 and 25.3 min) to afford 0.00035 mmol (6% yield)
of 4b. The purified product was lyophilized and stored at -75 °C.
MS calcd for C42H58N11O18P2S (M+) 1098, found 1098 (FAB, 3:1
glycerol/thioglycerol).
thioglycerol).
Gly
L-5-Hydroxytryptophanyl-tRNA -dCA (2e). Aminoacyl di-
nucleotide 2d was ligated to tRNA -COH as described for 1b, with
CUA
Gly
CUA
the following modification. The ligation reaction was incubated for
20 min at 37 °C. The precipitated material was resuspended, quantified,
and diluted to 1 µg/µL with 10 mM AcOH. The Nvoc group was
removed essentially as described.27 The diluted sample was irradiated
for 30 min at 4 °C, lyophilized to dryness, and stored at -75 °C until
use.
N-(Boc)-D,L-7-azatryptophan (3a). Amino acid 3a was prepared
as described by Rich et al., with minor modifications.10 To a stirred
solution of 502 mg (2.25 mmol) of 3 in 2.25 mL of a 2:1 H2O/dioxane
mixture at ambient temperature was added 0.5 mL (3.61 mmol) of
triethylamine. To the stirring solution was added 582 mg (2.67 mmol)
of di-tert-butyldicarbonate in 0.75 mL of dioxane. The resultant thick,
milky paste was stirred vigorously and became clear after ca. 40 min.
The reaction was stirred for 5.5 h and was worked up as described to
yield 99.6 mg (72% yield) of 3a as a white powder. Spectra were
identical to those reported.
EN-(dansyl)-L-lysinyl-tRNACGUlyA-dCA (4c). Aminoacyl tRNA 4c
was prepared as described for 1b.
S30 Lysate Reactions for the Synthesis of â-Galactosidase.
Plasmids pT7lac and pT7lac-7amb were purified by standard methods.5a
The S30 extract for in Vitro protein production was prepared with minor
modifications to the published procedure.5a The in Vitro wild-type and
suppression reactions were run according to established procedures.5a
Purification of â-Galactosidase. Purification of wild-type and
modified â-galactosidase was based on a protocol for purification of
the enzyme from E. coli and B. megaterium.28 The minicolumns
5′-O-Phosphoryl-2′-deoxycytidylyl-(3′-5′)-2′(3′)-O-[N-(Boc)-D,L-
7-azatryptophanyl]adenosine (3b). To a stirred solution of 80.6 mg
(0.36 mmol) of 3a in 0.5 mL of dry CH3CN at ambient temperature
was added 75.6 mg (0.47 mmol) of carbonyldiimidazole. After 20 min,
20.2 mg (0.032 mmol) of pdCpA in 0.5 mL of 100 mM NaH2PO4 was
(27) Ellman, J.; Mendel, D.; Anthony-Cahill, S.; Noren, C. J. Methods
Enzymol. 1991, 202, 301-336.
(28) Steers Jr., E.; Cuatrecasas, P. Methods Enzymol. 1974, 34, 350-
358.

Fluorescent Spectroscopy of â-Galactosidase
J. Am. Chem. Soc., Vol. 119, No. 1, 1997 11
consisted of disposable 1 mL poly(propylene) syringe barrels plugged
with 100% acrylic yarn and containing a luer-lock stopcock. The
columns were loaded with 100 µL of the suspended affinity resin (ca.
50 µL packed volume)29 and were then packed and equilibrated with
buffer A (50 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100
mM â-mercaptoethanol) by gravity flow at 4 °C. The lysate reactions
were diluted 2-fold with buffer A and loaded onto the pre-equilibrated
columns. The columns were washed extensively with buffer A until
the background A280 reading returned to baseline.30 The â-galactosidase
was eluted with 0.1 M sodium borate [pH 10.0 (200-500 µL)]. One
50 µL Phe suppression reaction was purified, and seven 50 µL dnsLys
suppression reactions were combined and purified.
As a control for hydrophobic binding of ꢀ-dnsLys, wild-type
â-galactosidase was incubated with the free amino acid 4 and then was
purified. Wild-type â-galactosidase from the crude lysate reaction (0.6
µg, 11 µL) was diluted to 350 µL (14.8 nM) with buffer A,
corresponding to the approximate concentration of dnsLys-â-galac-
tosidase produced in seven combined 50 µL lysate reactions (14.6 nM
assuming 0.085 µg/50 µL lysate reaction). Then ꢀ-dnsLys (4) was
added to a final concentration of 19.0 µM, corresponding to the
concentration that would be present if 100% of 4c hydrolyzed within
the lysate reaction. The â-galactosidase/ꢀ-dnsLys mixture was incu-
bated at 37 °C for 10 min, then was diluted 2-fold with buffer A and
purified as described above.
â-Galactosidase Assay. The 50 µL S30 lysate reactions were
quenched by addition of 750 µL of Z buffer (60 mM Na2HPO4, 40
mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM â-mercaptoethanol).
The reaction mixtures were then vortexed to suspend insoluble material,
and the desired amount was removed and diluted to 800 µL with Z
buffer.31 The diluted reaction mixture was equilibrated in a shaking
H2O bath at 37 °C. The â-galactosidase assay was initiated by addition
of 200 µL of ONPG (4 mg/mL in 60 mM Na2HPO4, 40 mM NaH2-
PO4, pre-equilibrated at 37 °C), followed by immediate vortexing and
incubation at 37 °C. The time that ONPG was added was recorded.
Upon the appearance of yellow color the assay was terminated by
addition of 500 µL of 1 M Na2CO3 and immediate vortexing. The
time of quenching was recorded. Assay mixtures were clarified by
centrifugation at 12000g for 10 min at 4 °C, and the absorbance at 420
nm (A420) was determined.
In order to quantify the assays, the A420 reading was divided by the
picomolar extinction coefficient of ONP (4.5 × 10-9 pM-1 cm-1) and
divided again by the pathlength (1 cm) to yield the picomolar
concentration of ONP. The pM concentration of ONP was multiplied
by the final assay volume (1.5 × 10-3 L), divided by the assay time
(min), and multiplied by the amount the 800 µL quenched reaction
was diluted22 to yield picomoles of ONP produced per minute per 50
µL S30 reaction (pmol of ONP/(min/50 µL S30 reaction)). In order
to convert pmol of ONP/min to µg of â-galactosidase, it was assumed
that the enzyme has the specific activity of pure â-galactosidase. The
specific activity of pure â-galactosidase is ca. 300 000 pmol of ONP/
(min/µg of â-galactosidase) at 28 °C,32 and the enzyme is ca. 1.4-fold
more active at 37 °C.33 Thus pmol of ONP/min was divided by 1.4
and then divided again by 300 000 pmol of ONP/(min/µg) to yield µg
of â-galactosidase/50 µL S30 reaction.
Fluorescence Analysis. The fluorescence emission spectra in Figure
1 were recorded with the Hitachi-4500. Excitation and emission slit
widths were 10 and 20 nm, respectively, and the PMT voltage was
950 V. Emission was scanned from 350 to 750 nm at a scan rate of
240 nm/min. A background spectrum of 1:2 Z buffer/0.1 M sodium
borate (pH 10.0) showed no significant emission.
If necessary, purified â-galactosidase samples eluting from the
columns were diluted to 350 µL with Z buffer. The fluorescence spectra
were then recorded. The concentrations of the samples were determined
on the basis of enzymatic activity by removing 50 µL from the
fluorescence sample, diluting with 750 µL of Z buffer, and assaying
for â-galactosidase activity as described above.
Acknowledgment. This work was supported by NIH Grant
GM42708 (A.R.C.) and in part by NIH Grant GM39750
(J.B.A.R.). L.E.S. is grateful to the NIH for support in the form
of an NRSA predoctoral training grant (GM07311). We thank
Dr. Monica Ryde´n-Aulin (Stockholm University) for supplying
E. coli strain US477, Dr. John Keener (UCI) for the pT7lac
plasmid and helpful advice regarding the preparation of S30
extracts, and Dr. G. Schwartz for advice regarding 5-OHTrp
chemistry.
(29) Binding capacity reported by Sigma: 2-8 mg of â-galactosidase/
mL of resin.
(30) Background readings normally returned to baseline after washing
with 2 mL of buffer; however, each column was washed with a minimum
of 5 mL.
JA963023F
(31) The assay can be run on the 800 µL quenched reaction; however,
in order for the assay to be accurate, the quenched reaction should be diluted
such that the formation of yellow color resulting from hydrolysis of ONPG
should take at least 10 min. Thus high yielding reactions must be diluted
several-fold. The amount of dilution is determined experimentally but is
generally within the 2-100-fold range (400-8 µL, respectively, of the
quenched reaction).
(32) One unit of â-galactosidase is defined as the amount of enzyme
that will hydrolyze 10-9 mol of ONPG/min at 28 °C. Pure â-galactosidase
has an activity of ca. 300 000 units/mg. See ref 14.
(33) Determined experimentally; data not reported. Since the S30 lysate
reactions were equilibrated at 37 °C, the assay reactions were run at 37 °C
for convenience.