(Received for publication, October 11, 1995)
From the
Human adenovirus contains a virion-associated proteinase
activity essential for the development of infectious virus. Maximal
proteinase activity in vitro had been shown to require three
viral components: the L3 23-kDa protein, an 11-amino acid cofactor
(pVIc), and the viral DNA. Here, we present a quantitative purification
procedure for a recombinant L3 23-kDa protein (recombinant
endoproteinase (rEP)) expressed in Escherichia coli and the
procedure that led to the purification and identification of pVIc as a
cofactor. The cofactors stimulate proteinase activity not by decreasing K, which changes by no more than 2-fold,
but by increasing k
. rEP alone had a small
amount of activity, the k
of which increased
355-fold with pVIc and 6072-fold with adenovirus serotype 2 (Ad2) DNA
as well. Curves of V
of rEP
pVIc complexes
with the substrate (Leu-Arg-Gly-Gly-NH)
-rhodamine as a
function of pH in the absence and presence of Ad2 DNA indicate that the
pK
values of amino acids that affect
catalysis are quite different from those that affect catalysis by the
cysteine proteinase papain. The pK
values
in the absence of Ad2 DNA are 5.2, 6.4, 6.9, 7.5, and 9.4, and those in
its presence are 5.2, 6.5, 7.4, and 8.8.
For many animal and plant viruses, a virus-coded proteinase
activity is vital for the synthesis of infectious virus (for review,
see (1) ). These virus-coded proteinases are appealing targets
for antiviral therapy. Human adenoviruses encode a proteinase activity
that is required for the maturation of infectious virions. Of the 12
major polypeptides from which adenovirus virions are assembled, six are
proteolytically processed. Weber (2) isolated a
temperature-sensitive mutant, H2ts-1 (ts-1), ()of human
adenovirus serotype 2 (Ad2) that lacks proteinase activity at the
nonpermissive temperature. Virions of ts-1 assemble at the
nonpermissive temperature, but contain precursors in place of the
mature components present in wild-type virus. Such immature virions
attach to cells, but fail to initiate a productive
infection(3, 4) . The mutation in ts-1 was identified
as a single base pair change in a 204-codon open reading frame (L3
23-kDa protein) at the 3`-end of the L3 family of late
messages(5) . The nucleotides in the L3 23-kDa open reading
frame were cloned into plasmids that permitted efficient expression in Escherichia coli(6) .
Recently, we developed a
specific, sensitive, and quantitative assay for the adenovirus
proteinase and used it to characterize the activity in disrupted
wild-type virus. ()The assay is based upon the observation
that the adenovirus proteinase will cleave small peptides with
sequences that correspond to the sequences on the amino-terminal side
of the cleavage sites in virion precursor proteins. For example, the
substrate (Leu-Arg-Gly-Gly-NH)
-rhodamine is cleaved to
Leu-Arg-Gly-Gly-NH-rhodamine by the adenovirus proteinase; this is
accompanied by a 3500-fold increase in fluorescence that is
proportional to the amount of proteinase.
We had previously shown
that when wild-type Ad2 virus was incubated with
(Leu-Arg-Gly-Gly-NH)-rhodamine, significant hydrolysis of
the substrate was observed, and that when ts-1 virus was incubated with
(Leu-Arg-Gly-Gly-NH)
-rhodamine, no hydrolysis of the
substrate was observed(7) . Little or no hydrolysis was
observed with purified recombinant L3 23-kDa protein (recombinant
endoproteinase (rEP)) expressed in E. coli. However, when ts-1
virus and rEP were incubated together with
(Leu-Arg-Gly-Gly-NH)
-rhodamine, significant hydrolysis of
the substrate occurred. This implied that cofactors may be required for
maximal activity. The first cofactor we discovered was the viral DNA.
If disrupted wild-type virus is treated with DNase, proteinase activity
is lost, but can be restored upon addition of Ad2 DNA(7) . A
second cofactor was shown to be a plasmin-sensitive virion protein (7) that turned out to be the 11-amino acid peptide from the C
terminus of the precursor to virion protein VI,
pVIc(7, 8) .
Here, we present our purification
procedure for a recombinant L3 23-kDa protein expressed in E.
coli. Although others have published purification procedures for a
recombinant form of the L3 23-kDa protein from E. coli(9, 10) and insect cells(8) , none of the
purification procedures utilized a quantitative assay, so there is, for
example, no report of increases in specific activity or even yields. We
also present our procedure for the purification and identification of
pVIc as a cofactor for proteinase activity. We then show that the
cofactors stimulate proteinase activity not by decreasing K, which changes by no more than 2-fold,
but by increasing k
, which increases
>6000-fold. By measuring initial velocities as a function of pH, we
show that the enzyme activity is clearly different from that of papain.
Moreover, the pK
values of the amino
acids that affect catalysis are different in the presence and absence
of Ad2 DNA.
Protein concentration was determined by the bicinchoninic acid
protein assay (Pierce) and/or for rEP with a calculated molar
absorbance coefficient at 280 nm of 26,510(11) . The
concentration of pVIc was determined by titration of its cysteine
residue with Ellman's reagent and confirmed by quantitative amino
acid analysis. The cysteine titration was done by adding 10 µl of
pVIc stock solution to 0.99 ml of Ellman's buffer (0.1 M
NaHPO
(pH 7.3) and 1 mM EDTA
containing 0.33 mM 5,5`-dithiobis(2-nitrobenzoic acid)) and
then monitoring the increase in absorbance at 412 nm. The moles SH/mol
of pVIc was calculated using a molar extinction coefficient at 412 nm
of 14,150 (12) for thionitrobenzoate.
Figure 1:
Purification of
adenovirus rEP. Samples of lysates from bacteria induced with IPTG to
express rEP (lane a), the 10,000 g supernatant of the lysate (lane b), the flow-through
fraction from the DEAE chromatography step (lane c), the rEP
activity pool from the S-Sepharose chromatography step (lane
d), rEP purified by passage over a zinc-iminodiacetic
acid-Sepharose column (lane e), and molecular mass markers (lane f) were separated by SDS-PAGE and stained with Coomassie
Brilliant Blue.
The lysate
was clarified by centrifugation at 10,000 g for 10
min. This fraction, named the supernatant, was loaded onto a 2.5
25-cm TSK-DEAE column equilibrated in 50 mM Tris (pH
8.0), 15 mM NaCl, and 5 mM
-mercaptoethanol. The
column was washed with 300 ml of the equilibration buffer. rEP was
located in the flow-through fractions by SDS-PAGE and by activity
assays. The fractions with rEP were pooled and named DEAE FT.
The
DEAE FT pool was fractionated on a 1.6 15-cm S-Sepharose Fast
Flow column equilibrated in 50 mM Tris (pH 8.0), 15 mM NaCl, and 1 mM DTT. After loading, the column was washed
with 200 ml of 50 mM Tris (pH 8.0), 15 mM NaCl, and 1
mM DTT to remove the Triton X-100. Then, a 150-ml linear
gradient of 15-400 mM NaCl in 50 mM Tris (pH
8.0) was applied to the column. The fractions containing rEP were
identified and pooled and named SSEPH. rEP consistently eluted from
this column at an ionic strength of 0.1 M NaCl.
The SSEPH
pool was applied to a 1.6 5-cm chelating Sepharose column
charged with zinc. Charging with zinc was accomplished by a thorough
washing of the resin with 0.05 M EDTA and 1 M NaCl,
followed by washing with water to remove the EDTA, and then followed by
washing with 0.2 M ZnCl
in 5 mM HCl.
Next, the column was equilibrated in 25 mM HEPES (pH 8.0) and
0.1 M NaCl. The column was loaded with the SSEPH pool and
washed with the equilibration buffer until the absorbance at 280 nm was
<0.02. Then, a series of solutions were applied in steps, each step
containing 25 mM HEPES (pH 8.0) and the indicated
constituents: 1 M NaCl; 0.1 M NaCl; 35 mM imidazole and 0.1 M NaCl; 0.1 M NaCl; 35
mM imidazole and 0.1 M NaCl; 0.1 M NaCl; 35
mM imidazole and 0.1 M NaCl; and 0.1 M NaCl.
rEP eluted in 25 mM HEPES (pH 8.0), 0.1 M NaCl, and
0.01 M EDTA. After dialysis against 20 mM HEPES (pH
8.0), 5 mM NaCl, and 0.1 mM EDTA, the purified enzyme
was stored at -70 °C.
The flow-through fraction from the second Centricon-30
centrifugation was evaporated to dryness, dissolved in 0.1%
trifluoroacetic acid, and applied to a C column (Aquapore
OD-300 7µ, 2.1
100 mm). The activity was eluted by a linear
gradient of 0-30% acetonitrile in 0.1% trifluoroacetic acid at a
rate of 1%/min. Assays of fractions from each peak were performed in
the presence of Ad2 DNA and rEP.
Figure 2:
Processing of Ad2 ts-1 precursor proteins
by rEP. Twice banded Ad2 ts-1 virions were disrupted by two cycles of
freeze/thaw followed by heat treatment. Reactions of 0.18 ml contained
10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM octyl glucoside, 3 10
virions, and 0.25
µM rEP. After the indicated times at 37 °C, aliquots
were removed from the reactions, and the proteins were fractionated by
SDS-PAGE on 8-25% gradient gels. Lanes a and h,
wild-type Ad2 virions; lane g, Ad2 ts-1 virions; lanes
b-f, Ad2 ts-1 virions incubated with rEP for 6, 4, 2, 1, and
0.5 h, respectively. The precursor proteins of the ts-1 virion and
their mature counterparts in the wild-type (wt) Ad2 virion are
labeled. The proteins were visualized by silver
staining.
Figure 3: Initial purification of the second cofactor activity. Virus was disrupted by treatment with 10% pyridine. The pellet and supernatant (Sup) were obtained after centrifuging disrupted virus. The pellet was solubilized, placed in a Centricon-30, and centrifuged to yield retentate-1 and flow-through-1. Ammonium acetate to 1 M was added to retentate-1, and the solution was recentrifuged to yield retentate-2 and flow-through-2. Assays were performed in the presence of 3 nM rEP and in the absence or presence of 778 ng/ml Ad2 DNA.
The final step in the purification of the second cofactor was
reverse-phase chromatography. The flow-through fraction from the second
Centricon-30 centrifugation was applied to a C column, and
the cofactor activity was eluted with a linear gradient of 0-30%
acetonitrile (Fig. 4). Each peak from the column was assayed for
proteinase activity. Only the three peaks marked with arrows showed proteinase activity in the presence of rEP and Ad2 DNA. A
profile of the proteinase activity in each of the three peaks is shown
in the inset in Fig. 4. Of the total cofactor activity
applied to the C
column, 7% was recovered in peak a, 22%
in peak b, and 14% in peak c. The final yield was >49%.
Figure 4:
Final step in the purification of the
second cofactor activity. The flow-through fraction from the second
Centricon-30 centrifugation was applied to a C column, and
the activity was eluted by a linear gradient of 0-30%
acetonitrile in 0.1% trifluoroacetic acid. Inset, assays of
fractions from peaks a, b, and c were performed in the presence of 778
ng/ml Ad2 DNA and 2.2 nM rEP. mAU, milli-absorbance
units.
Figure 5: Amino acid sequence of the second cofactor and comparison with the amino acid sequence of the precursor to adenovirus protein VI. The amino acid sequences of the proteins in peaks a-c in Fig. 4(inset) were determined in a gas-phase sequencer. The question mark at position 6 indicates a variable yield of lysine, as described under ``Results.'' The amino acid at position 10 was presumed to be a cysteine. The two adenovirus proteinase consensus cleavage sequences in pVI are underlined, and the location of the second cofactor sequence beginning at position 240 is in boldface. The amino acid sequence of pVI was from Roberts et al.(14) .
The proteins in peaks a-c of Fig. 4were
also subjected to time-of-flight mass spectrometry. In peak a, there
was one major species with an M of 1350. Thus,
there was mostly pVIc present. In peak b, there were two species
present at a ratio of 2:1, one with an M
of 1350
and the other with an M
of 2700. Thus, there was
mostly pVIc present, but also some pVIc dimer. In peak c, there was one
major species with an M
of 1350.
Figure 6: Reconstitution of proteinase activity in vitro with purified components and titration with the second cofactor (A) and with Ad2 DNA (B). A, assays in 400 µl contained 1.42 µg/ml Ad2 DNA, either 2 nM (open circles) or 4 nM (closed circles) rEP, and the indicated volumes of cofactor activity purified as described in the legend to Fig. 3. B, assays in 400 µl contained 2 nM rEP, 10 µl of cofactor activity purified as described in the legend to Fig. 3, and the indicated concentrations of Ad2 DNA. Rates are expressed as the difference in rates between assays in the presence and absence of the cofactor (A) and Ad2 DNA (B).
Figure 7:
Optimization of assay conditions in the
absence (closed circles) and presence (open circles)
of Ad2 DNA: temperature (A), ionic strength (B),
octyl glucoside (C), and dithiothreitol (D). In A, complexes between rEP and pVIc were formed by incubating 70
nM rEP and 208 nM pVIc in 0.9 ml of 0.1 mM TAPS (pH 8.5), 10 mM octyl glucoside, 1 mM EDTA,
and 0.5 mM DTT for 5 min at 37 °C. The rEPpVIc
complexes were then assayed at pH 8.5 using TAPS buffer. Complexes
between rEP, pVIc, and Ad2 DNA were formed the same way, except that
the reactions contained 14 nM rEP, 200 nM pVIc, and
140 ng/ml Ad2 DNA. The rEP
pVIc
Ad2 DNA complexes were
assayed at pH 8.0 using Tris buffer. After the preincubations, 0.1 ml
of 0.1 M buffer, 30 µM
(Leu-Arg-Gly-Gly-NH)
-rhodamine, 10 mM octyl
glucoside, 1 mM EDTA, and 0.5 mM DTT was added; the
reactions were incubated at the indicated temperatures; and after 10
min, the increase in fluorescence was determined. In B-D, complexes were formed in 0.9 ml as described for A, except for the absence of the indicated variable. Then, 0.1
ml was added as described for A, except that it contained 10
times the final concentration of the indicated variable, and the
increase in fluorescence at 37 °C was monitored as a function of
time.
Based upon these and other observations, standard assay
conditions were adopted that included 37 °C, 10 mM buffer
at pH 8.5 for the absence of DNA and at pH 8.0 for the presence of DNA,
and 10 mM octyl glucoside. Occasionally, a preparation of rEP
was stimulated by DTT and/or EDTA, in which case, assays also included
0.5 mM DTT and/or 1 mM EDTA. EDTA at higher
concentrations was inhibitory; with rEPpVIc complexes,
half-maximal activity was lost at 50 mM EDTA (data not shown).
Under standard assay conditions, the increase in fluorescence as a
function of time with (Leu-Arg-Gly-Gly-NH)
-rhodamine as the
substrate was linear for >30 min.
Figure 8:
Maximal velocity as a function of pH for
rEPpVIc complexes (A) and for rEP
pVIc
Ad2 DNA
complexes (B). Complexes were formed by incubating 63 nM rEP and 200 nM pVIc (A) or 15.75 nM rEP, 200 nM pVIc, and 5.6 pM Ad2 DNA (B) for 5 min at 37 °C in 0.9 ml of 0.1 mM TAPS
(pH 8.5) containing 1.1 mM EDTA, 0.55 mM DTT, and 11
mM octyl glucoside. Then, 0.1 ml was added containing
components such that the final concentration of buffer was 10 mM and that of (Leu-Arg-Gly-Gly-NH)
-rhodamine was 3
µM, and the ionic strength was 16 mM. The
increase in fluorescence was then measured as a function of time. The
buffers used were sodium citrate (pH 4.0 and 4.6), sodium acetate (pH
5.0 and 5.5), MES (pH 6), sodium cacodylate (pH 6.0-6.8), HEPES
(pH 7-7.8), Tris (pH 8.0 and 8.5), CHES (pH 9.0 and 9.5), and
CAPS (pH 10.0 and 10.5). The curves were fitted using the program
Peakfit (Jandel Scientific) assuming merged gaussian peaks (dotted
lines).
The rEP protein was purified to apparent homogeneity using
three chromatographic steps with an overall yield of 66%. About 125 mg
of rEP were induced by IPTG in a 4-liter culture of cells grown to an
absorbance at 600 nm between 0.5 and 0.6 before induction by IPTG. rEP
does not bind to DEAE probably because of its high isoelectric point,
which was calculated to be 8.68. Also, the DEAE step was used to remove
nucleic acids because they bind to the column at NaCl concentrations
<0.3 M(15) . The S-Sepharose anion-exchange column
gave the largest increase in specific activity, from 67.7 to 485
units/mg. A chelating Sepharose column charged with zinc was used
because rEP contains eight free cysteines ()and three
histidines. rEP bound quite tightly to this column as it had to be
eluted with EDTA. The final step in the purification of rEP was
dialysis against 0.5 mM EDTA. This was done to remove all
traces of zinc, which is an inhibitor of enzyme activity. With
(Leu-Arg-Gly-Gly-NH)
-rhodamine as the substrate, the
specific activity increased from 22.4 to 815 nmol of substrate
hydrolyzed per s/mg of protein.
We were able to observe the processing of most of the virion precursor proteins by adding purified rEP to disrupted ts-1 virus. This established that rEP can find its cofactors and become activated and that activated rEP can cleave in vivo substrates. Furthermore, this indicated that rEP need not be post-translationally modified, e.g. glycosylated, to become activated and cleave in vivo substrates, unless such modifying enzymes and their substrates are in the virion. These data also allow one to conclude that the gene coding for the L3 23-kDa protein is indeed the gene whose inactivation gives rise to the ts-1 phenotype. The viral DNA was implicated as a cofactor because pretreatment of disrupted ts-1 virus with DNase prevented processing after the addition of rEP.
The purification of the second cofactor, pVIc, was very difficult, and, with hindsight, we now know why. We lost activity upon column chromatography because it was so basic, it stuck to glass. We lost activity upon dialysis because it was so small, it passed through the pores in the tubing. Consistent with the second cofactor being a small protein are the observations that boiling for 5 min did not irreversibly denature it; at high ionic strength, it passed through a Centricon-3 (3000-Da cutoff); and when incubated in 5 M urea, it rapidly regained activity upon dilution of the urea (data not shown). The amino acid sequence of the second cofactor is consistent with the biochemical data. The second cofactor was sensitive to plasmin because it contains one lysine and three arginines.
The purification data in Fig. 3implied that pVIc was bound to the viral DNA. At each step before the second Centricon-30 centrifugation, cofactor activity was not greatly stimulated by the addition of Ad2 DNA, but after, enzyme activity was greatly stimulated by the addition of Ad2 DNA. The second cofactor was not in the flow-through fraction after the first Centricon-30 centrifugation because it is a very basic protein that was probably bound to the viral DNA. High ionic strength would dissociate it from the viral DNA, and thus after the second Centricon-30 centrifugation, it was in the flow-through fraction. Cofactor activity in the flow-through fraction was greatly stimulated by the addition of Ad2 DNA.
The flow-through fraction from the
Centricon-30 centrifugation gave numerous peaks on a reverse-phase
C column. Three of the peaks contained cofactor activity.
Sequencing of the three peaks indicated that there was a variable yield
in lysine at position 6 and no amino acid was detected at position 10,
where we expected a cysteine. Time-of-flight mass spectrometry analysis
indicated that the major species in each peak had an M
of 1350, consistent with the presence of a monomer of pVIc.
Webster et al.(8) purified the cofactor by
solubilizing virions in 4 M guanidine HCl and fractionating by
fast protein liquid chromatography on a Superdex S-75 gel filtration
column. Two peaks of complementing activity were detected by subsequent
reverse-phase high pressure liquid chromatography. One peak was the
monomer of pVIc, and the other peak was the disulfide dimer of pVIc.
We were able to reconstitute maximal proteinase activity with
purified components: rEP, pVIc purified from wild-type virus, and Ad2
DNA. The cofactors stimulated proteinase activity by increasing k >6000-fold. The K
changed by less than a factor of 3. Previous in vitro assays for the Ad2 proteinase activity were successful because
they utilized Ad2 precursor proteins in an extract from ts-1-infected
cells as substrate and disrupted wild-type virus (16) or rEP as
the source of proteinase (6) or synthetic peptides as substrate
and disrupted wild-type virus as the
proteinase(17, 18) . Hence, in those assays, both
cofactors were present.
Other proteinases require cofactors for
activity, but none, so far, exhibits the requirements of the Ad2
proteinase. Several neutral proteinases need Ca for
activity(19) . Some proteinases utilize ATP(20) . A
serine proteinase anchored to the membrane of Plasmodium falciparum by a covalently attached glycosylphosphatidylinositol moiety is
activated by phosphatidylinositol-specific phospholipase
C(21) . Many proteinases are synthesized as zymogens and must
be activated by proteolytic cleavage, e.g. the activation of
trypsinogen to trypsin by enterokinase or trypsin (22) or the
activation of plasminogen to plasmin by urokinase(23) . The
assembly and activation of some of the proteins of the blood
coagulation system require a negatively charged surface(24) .
The virus-coded proteinases from the human immunodeficiency virus and
avian sarcoma/leukosis viruses require themselves as cofactors as
homodimers are the active form(25, 26, 27) .
Although the E. coli RecA protein can facilitate the cleavage
of the LexA protein bound to DNA, RecA apparently does so as an
allosteric effector and not as a proteinase with an active-site
nucleophile(28) .
The experiments on optimizing the assay
conditions for the proteinase activity of rEPpVIc complexes in
the absence and presence of the viral DNA revealed an unusual
sensitivity to ionic strength. In the absence of the DNA, 10 mM NaCl inhibited 50% of the enzyme activity. In the presence of Ad2
DNA, 45 mM NaCl inhibited 50% of the enzyme activity. In
contrast to these results, in disrupted virions, 300 mM NaCl
was required for 50% inhibition of activity.
The latter
experiment implied that direct inhibition either of the binding of
substrate to the active site or, once bound, of the rate of catalysis
probably occurs at NaCl concentrations closer to 300 mM than
to 30 mM. Thus, NaCl concentrations of 10-45 mM must inhibit enzyme activity by interfering with formation of an
active complex, a complex already formed in a disrupted virus particle.
Although we settled upon standard assay conditions, occasionally they must be altered. We have found (data not shown) that with aged disrupted virus, when compared with newly isolated, disrupted virus, the degree of stimulation by low concentrations of DTT varied from zero with newly isolated, disrupted virus to 4-5-fold with aged disrupted virus. Purified rEP exhibits a similar pattern in that as it ages during storage, 0.5 mM DTT will stimulate more and more activity. This result can be interpreted as signifying the importance of the oxidation states of certain cysteine residues. Similarly, the presence of EDTA at concentrations <2 mM sometimes stimulated proteinase activity (data not shown). Perhaps some zinc, which is a potent inhibitor of enzyme activity, remained with the rEP after chromatography on a chelating Sepharose column charged with zinc.
The nature of the active site of the Ad2 proteinase is unclear. The
inhibitor profile of wild-type virus does not correspond to profiles
exhibited by classical serine or cysteine
proteinases(17, 18, 29, 30) . Examination of the L3 23-kDa gene sequence led Webster et al.(17, 18) to propose that the Ad2 proteinase may
be a member of a new subclass of cysteine proteinases described by
Brenner(31) , by Bazan and Fletterick(32) , and by
Gorbalenya et al.(33) . Based upon site-directed
mutagenesis studies, two groups have argued that the enzyme is a
cysteine proteinase and that Cys-104 is the active-site nucleophile (34, 35) .
The requirement for DNA as a cofactor
for a proteinase activity is unprecedented. It is clearly required in
the Ad2 virion because proteinase activity is lost upon treatment with
DNase and restored upon addition of Ad2 DNA. In addition, the precursor
proteins in disrupted ts-1 virus are processed upon incubation with
rEP. However, no processing occurs if disrupted virions are pretreated
with DNase. Reconstitution of proteinase activity in vitro with purified components indicates that Ad2 DNA affects k and not K
. Webster et
al.(36) found no stimulation of rEP
pVIc complex
activity by Ad2 DNA.
The experiments on V as
a function of pH with rEP
pVIc complexes in the absence and
presence of Ad2 DNA indicated that the enzyme is quite different from
the cysteine proteinase papain. Papain contains an active-site
thiolate-imidazolium ion pair between His-159 and Cys-25(37) .
The second-order acylation rate constant (k
/K
) as a function of pH
conforms to a bell-shaped curve. The two ionizing groups with
pK
values near 4 and 8.5 probably correspond to
His-159 and Cys-25, respectively, more appropriately to the formation
and decomposition of the ion pair. An active-site thiolate-imidazolium
ion pair in the adenovirus proteinase could have pK
values in the absence of Ad2 DNA of 5.17 and 9.43, whereas in the
presence of Ad2 DNA, the pK
values could be 5.15
and 8.78. These pK
values are similar to the
normal pK
values of 6.0 for histidine and 8.3 for
cysteine. Our thiol protection experiment at pH 5.0 in vivo with disrupted virus is consistent with the presence of a
thiolate-imidazolium ion pair.
The experiments on V as a function of pH implied that rEP
pVIc
complexes bind to Ad2 DNA. The profiles of rEP
pVIc complexes in
the absence and presence of Ad2 DNA are different: pK
values of 5.2, 6.4, 6.9, 7.5, and 9.4 versus 5.2, 6.5,
7.4, and 8.8, respectively. This indicates that Ad2 DNA does affect the
pK
values of some of the amino acids involved in
catalysis and therefore implies that the rEP
pVIc complexes bind
to Ad2 DNA. In addition, the results of measuring the V
of rEP
pVIc complexes in the presence of
Ad2 DNA are similar to those obtained with enzyme activity in disrupted
virus. The pK
values for disrupted virus are 5.2,
6.2, 7.2, and 8.4. This implied that in the virion, rEP
pVIc
complexes are bound to the viral DNA.