Influence of substrate structure on the catalytic efficiency of hydroxysteroid sulfotransferase STa in the sulfation of alcohols

Article abstract of DOI:10.1021/tx950065t

Sulfotransferase a (STa) is an isoform of hydroxysteroid (alcohol) sulfotransferase that catalyzes the formation of sulfuric acid esters from both endogenous and xenobiotic alcohols. Among its various functions in toxicology, STa is the major form of hepatic sulfotransferase in the rat that catalyzes the formation of genotoxic and carcinogenic sulfuric acid esters from hydroxymethyl polycyclic aromatic hydrocarbons. The goal of the present study was to elucidate fundamental quantitative relationships between substrate structure and catalytic activity of STa that would be applicable to these and other xenobiotics. We have modified previous procedures for purification of STa in order to obtain sufficient amounts of homogeneous enzyme for determination of k(cat)/K(m) values, a quantitative measure of catalytic efficiency. We determined the catalytic efficiency of STa with benzyl alcohol and eight benzylic alcohols that were substituted with n- alkyl groups (C(n)H(2n+1), where n = 1-8) in the para position, and the optimum value for k(cat)/K(m) in these reactions was obtained with n- pentylbenzyl alcohol. Correlations between logarithms of k(cat)/K(m) values and logarithms of partition coefficients revealed that hydrophobicity of the substrate was a major factor contributing to the catalytic efficiency of STa. Primary n-alkanols (C(n)H(2n+1)OH, where n = 3-16) exhibited an optimum k(cat)/K(m) for C₉-C₁₁ and a linear decrease in v(max) of the reaction for C₃-C₁₄; 15- and 16-carbon n-alkanols were not substrates for STa. These results indicated limits to the length of the extended carbon chain in substrates. Such limits may also apply to hydroxysteroids, since cholesterol was inactive as either substrate or inhibitor of STa. Furthermore, the importance of steric effects on the catalytic efficiency of STa was also evident with a series of linear, branched, and cyclic seven-carbon aliphatic alcohols. In conclusion, our results provide fundamental quantitative relationships between substrate structure and catalytic efficiency that yield insight into the specificity of STa for both endogenous and xenobiotic alcohols.

Full text of DOI:10.1021/tx950065t

Chem. Res. Toxicol. 1996, 9, 67-74
67
In flu en ce of Su bstr a te Str u ctu r e on th e Ca ta lytic
Efficien cy of Hyd r oxyster oid Su lfotr a n sfer a se STa in th e
Su lfa tion of Alcoh ols
Guangping Chen, Erden Banoglu, and Michael W. Duffel*
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of
Iowa, Iowa City, Iowa 52242
Received April 21, 1995X
Sulfotransferase a (STa) is an isoform of hydroxysteroid (alcohol) sulfotransferase that
catalyzes the formation of sulfuric acid esters from both endogenous and xenobiotic alcohols.
Among its various functions in toxicology, STa is the major form of hepatic sulfotransferase in
the rat that catalyzes the formation of genotoxic and carcinogenic sulfuric acid esters from
hydroxymethyl polycyclic aromatic hydrocarbons. The goal of the present study was to elucidate
fundamental quantitative relationships between substrate structure and catalytic activity of
STa that would be applicable to these and other xenobiotics. We have modified previous
procedures for purification of STa in order to obtain sufficient amounts of homogeneous enzyme
for determination of kcat/Km values, a quantitative measure of catalytic efficiency. We
determined the catalytic efficiency of STa with benzyl alcohol and eight benzylic alcohols that
were substituted with n-alkyl groups (CnH2n+1, where n ) 1-8) in the para position, and the
optimum value for kcat/Km in these reactions was obtained with n-pentylbenzyl alcohol.
Correlations between logarithms of kcat/Km values and logarithms of partition coefficients
revealed that hydrophobicity of the substrate was a major factor contributing to the catalytic
efficiency of STa. Primary n-alkanols (CnH2n+1OH, where n ) 3-16) exhibited an optimum
kcat/Km for C9-C11 and a linear decrease in vmax of the reaction for C3-C14; 15- and 16-carbon
n-alkanols were not substrates for STa. These results indicated limits to the length of the
extended carbon chain in substrates. Such limits may also apply to hydroxysteroids, since
cholesterol was inactive as either substrate or inhibitor of STa. Furthermore, the importance
of steric effects on the catalytic efficiency of STa was also evident with a series of linear,
branched, and cyclic seven-carbon aliphatic alcohols. In conclusion, our results provide
fundamental quantitative relationships between substrate structure and catalytic efficiency
that yield insight into the specificity of STa for both endogenous and xenobiotic alcohols.
for these reactions have led to the conclusion that the
hydroxysteroid sulfotransferases are the principal en-
zymes involved in the sulfation of these hydroxymethyl
polycyclic aromatic hydrocarbons (3, 7).
In tr od u ction
Hydroxysteroid sulfotransferases are key enzymes both
in the metabolism of endogenous hydroxysteroids such
as dehydroepiandrosterone (DHEA)1 and in the biotrans-
formation of xenobiotic alcohols with a diverse array of
chemical structures. Among xenobiotic alcohols, the
hydroxymethyl polycyclic aromatic hydrocarbons have
elicited particular toxicological interest. Many hydroxym-
ethyl polycyclic aromatic hydrocarbons are activated to
carcinogenic and/or mutagenic sulfuric acid esters through
reactions catalyzed by sulfotransferases. Representative
examples of these reactions include sulfation of 7-(hy-
droxymethyl)benz[a]anthracene (1), 7-(hydroxymethyl)-
12-methylbenz[a]anthracene (HMBA) (2), 5-(hydroxym-
ethyl)chrysene (3), 6-(hydroxymethyl)benzo[a]pyrene (4),
4-hydroxy- and 3,4-dihydroxy-3,4-dihydrocyclopenta[c,d]-
pyrene (5), 1-(hydroxymethyl)pyrene (6), and 9-(hydroxym-
ethyl)anthracene (6). Studies on species and sex differ-
ences, as well as differences in susceptibility to inhibitors,
The hydroxysteroid sulfotransferases were first puri-
fied to homogeneity by J akoby and co-workers, and the
substrate specificities of these enzymes led to their
designation as both hydroxysteroid and alcohol sul-
fotransferases (8-10). More recently, Watabe and co-
workers purified the major form of hydroxysteroid sul-
fotransferase in rat liver and found that it was active
with both hydroxysteroids and hydroxymethyl polycyclic
aromatic hydrocarbons (11, 12). These investigators
named the homogeneous enzyme as sulfotransferase a
(STa) and characterized the relative activities of the
enzyme with four hydroxymethyl polycyclic aromatic
hydrocarbons, each at a single substrate concentration
(11). STa has been cloned and sequenced (13), and rat
hepatic STa that is catalytically active with a hydroxym-
ethyl polycyclic aromatic hydrocarbon has recently been
expressed in Escherichia coli (14).
* To whom correspondence should be addressed. Telephone: (319)
335-8840; FAX: (319) 335-8766; E-mail on Internet: michael-
duffel@uiowa.edu.
While the first homogeneous preparations of hydrox-
ysteroid (alcohol) sulfotransferases were characterized as
generally prefering hydrophobic substrates with alcohol
functional groups (8-10), detailed structure-activity
studies leading to quantitation of optimal substrate
hydrophobicity and systematic determination of steric
effects on catalytic efficiency of these enzymes have not
X Abstract published in Advance ACS Abstracts, December 1, 1995.
1
Abbreviations: DHEA, dehydroepiandrosterone; HMBA, 7-(hy-
droxymethyl)-12-methylbenz[a]anthracene; kcat, catalytic turnover num-
ber (mol of sulfuric acid ester product formed/min/mol of STa subunit);
log P, logarithm of the partition coefficient; PAP, adenosine 3′,5′-
diphosphate; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; STa, hy-
droxysteroid (alcohol) sulfotransferase a.
0893-228x/96/2709-0067$12.00/0 © 1996 American Chemical Society
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68 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Chen et al.
spectroscopy (Nicolet Model 205). These data were identical
with literature values for the previously reported 4-alkyl
substituted benzylic alcohols: 4-propylbenzyl alcohol (21), 4-pen-
tylbenzyl alcohol (21), 4-hexylbenzyl alcohol (22), and 4-octyl-
benzyl alcohol (21). Spectroscopic data observed for 4-heptyl-
benzyl alcohol were as follows: NMR, in CD3OD, δ 0.89-1.6
ppm (m, 13 H, n-hexane), δ 2.5 ppm (t, 2H, -CH2C6H4-), δ 4.55
ppm (s, 2H, -C6H4CH2OH), and δ 7.1-7.3 ppm (dd, 4H, -C6H4-
); and IR (KBr) ν ) 3320 cm-1 (O-H stretch), 3050-3010 cm-1
(C-H stretch, aromatic), 3000-2820 cm-1 (C-H stretch, ali-
phatic), 1510 and 1460 cm-1 (C-C ring stretch), 1210 cm-1
(O-H bend), and 1010 cm-1 (O-H stretch, primary alcohol).
P a r tition Coefficien ts of Ben zylic Alcoh ols. Partition
coefficients were determined by allowing the benzylic alcohols
to partition between 8.0 mL of 0.25 M potassium phosphate (pH
7.0), that had been presaturated with 1-octanol, and 2.0 mL of
1-octanol, that had been presaturated with the phosphate buffer.
Each benzylic alcohol was dissolved in the aqueous phosphate
buffer, 1-octanol was added, and the mixture was gently shaken
in sealed glass tubes at 25 °C for 24 h. After centrifugation to
completely separate the phases, the final distribution of each
benzylic alcohol between the aqueous and organic layers was
determined by UV absorbance of the aqueous phase at 219 nm.
Partition coefficients were determined for at least three con-
centrations of each benzylic alcohol (chosen between 20% and
80% of the limit for solubility of each specific benzylic alcohol
in the aqueous buffer). All partition coefficients determined in
this manner were independent of the starting concentration of
benzylic alcohol in the aqueous phase.
been previously reported. In the present article, we
describe a series of investigations on the effects of
hydrophobicity and other characteristics of substrate
structure on the catalytic efficiency of STa in the sulfation
of alcohols. We have chosen STa for these studies due
to the opportunity to utilize a major isoform of hydrox-
ysteroid (alcohol) sulfotransferase for which a deduced
amino acid sequence is available (13). In addition to the
investigation on structure-activity relationships with
STa, we also present improvements in enzyme purifica-
tion that were essential to preparation of the quantities
of homogeneous and stable STa required for these stud-
ies.
Ma ter ia ls a n d Meth od s
Cau tion : 7-(Hydr oxymeth yl)-12-meth ylben z[a ]a n th r a cen e,
like oth er polycyclic a r om a tic h yd r oca r bon d er iva tives,
is a poten tia l h u m a n ca r cin ogen a n d sh ou ld be h a n d led
a ccor d in g to th e a ppr opr ia te NIH gu id elin es. Benzyl
alcohol, 4-methylbenzyl alcohol, 4-ethylbenzyl alcohol, 4-butyl-
benzyl alcohol, all aliphatic and alicyclic alcohols, all phenols,
4-propylbenzoic acid, 4-pentylbenzoic acid, 4-hexylbenzoic acid,
4-heptylbenzoic acid, and 4-octylbenzoic acid were obtained from
Aldrich Chemical Co. (Milwaukee, WI), and purity was con-
firmed by thin layer chromatography (TLC). 7-(Hydroxym-
ethyl)-12-methylbenz[a]anthracene was obtained from the NCI
Chemical Carcinogen Repository, Midwest Research Institute
(Kansas City, MO), and the chemical structure was verified by
UV and NMR spectroscopy. Phenols were purified by recrys-
tallization or redistillation before use. Dehydroepiandrosterone
(DHEA), cholesterol, estrone, adenosine 3′,5′-diphosphate (PAP),
DEAE-Sephadex A-50, and adenosine 3′,5′-diphosphate-linked
agarose (N-6 attachment with an 8-atom spacer) (PAP-agarose)
were obtained from Sigma Chemical Co. (St. Louis, MO).
Spectra/P or dialysis tubing with 12 000-14 000 molecular
weight cutoff was obtained from Spectrum Medical Industries
(Houston, TX). Bio-Gel HT hydroxylapatite and Tween-20 (EIA
grade) were obtained from Bio-Rad Laboratories (Hercules, CA).
3′-Phosphoadenosine 5′-phosphosulfate (PAPS) was prepared by
chemical synthesis (15). All other assay reagents and buffer
components were obtained from commercial sources.
Molecu la r Mod elin g. Space-filling models were created
using Sybyl software (Tripos Associates, St. Louis, MO) on an
Indigo workstation (Silicon Graphics, Mountain View, CA) at
the University of Iowa Image Analysis Facility. The energy of
each molecule was minimized using Maximin2 with the Tripos
Force Field.
Assa ys of Su lfotr a n sfer a se Activity d u r in g P u r ifica -
tion . Throughout the protein purification, sulfotransferase
activity was determined by the methylene blue paired ion
extraction assay described by Nose and Lipmann (16). Hydrox-
ysteroid sulfotransferase activity was determined in assay
mixtures containing 0.25 M sodium acetate (pH 5.5), 7.5 mM
2-mercaptoethanol, 0.2 mM PAPS, 50 µM DHEA, and 50 µL of
the appropriate column fraction in a total volume of 0.40 mL.
The reactions were started by addition of DHEA and were
incubated at 37 °C for 10-30 min. Determination of 4-nitro-
phenol sulfotransferase activity during protein purification was
carried out by the same general assay method, with the
exception that 0.25 M potassium phosphate (pH 7.0) was
substituted for sodium acetate, and 0.25 mM 4-nitrophenol
replaced DHEA. Enzyme units are expressed as nanomoles of
sulfuric acid ester product formed per minute. Protein concen-
trations were determined using a modified Lowry procedure
(17), with crystalline bovine serum albumin as standard.
Syn th esis of Ben zylic Alcoh ols. 4-Propyl- , 4-pentyl-,
4-hexyl-, 4-heptyl-, and 4-octylbenzyl alcohol were synthesized
by reduction of the corresponding 4-alkyl substituted benzoic
acids using the same general procedure (20) for each. Lithium
aluminum hydride (10 mL of a 1.0 M solution in diethyl ether)
was placed in a 50 mL three-necked flask equipped with a
dropping funnel and reflux condenser. The reaction vessel was
protected from moisture by calcium chloride drying tubes
attached to the openings. The reaction mixture was cooled in
an ice bath, and a 5 mL solution of anhydrous diethyl ether
containing 1 g of the appropriate benzoic acid was added at a
rate that produced a gentle reflux. the reaction mixture was
stirred in the ice bath for 1 h, then stirred at room temperature
for 1 h, and finally heated at reflux for 1 h. After cooling the
reaction mixture to 0 °C, water was cautiously added to
decompose the excess hydride. Following addition of 20 mL of
10% (v/v) sulfuric acid, the solution was extracted with four 50
mL portions of diethyl ether, and the combined organic layers
were dried over anhydrous sodium sulfate. The ether was
evaporated to yield the benzylic alcohol as an oily yellow residue,
which was subsequently purified by preparative silica gel
chromatography on a chromatotron with ethyl acetate/cyclo-
hexane (8:2) and chloroform/cyclohexane (9.9:0.1) as mobile
phases. Purity was confirmed by TLC on silica gel in three
solvent systems: ether, ethanol/ethyl acetate/cyclohexane (6:3:
1), and chloroform/n-hexane (9.9: 0.1). Yields of 50-60% were
obtained for each of the five benzylic alcohols prepared by this
procedure.
P u r ifica tion of STa . Hydroxysteroid sulfotransferase STa
was purified to homogeneity using a procedure incorporating
modifications to previously published (11, 12) procedures. Six
female Sprague-Dawley rats (9-10 weeks of age) were decapi-
tated, and the livers were removed and immediately placed in
ice-cold buffer A (50 mM Tris‚HCl (pH 7.5), 0.25 M sucrose, and
3 mM 2-mercaptoethanol). All subsequent purification steps
were conducted at 4 °C. The livers were homogenized in buffer
A (2:1 v/w, homogenate) in a Potter-Elvehjem homogenizer. The
homogenate was subjected to centrifugation at 100000g for 100
min, and the resulting supernatant fraction was filtered on a
double layer of cheesecloth.
Following addition of Tween-20 to a final concentration of
0.05% v/v, the 100000g supernatant fraction was applied to a
4.8 × 45 cm DEAE-Sephadex A-50 column that had been pre-
equilibrated with buffer B (buffer A + 0.05% v/v Tween-20). The
column was washed with 800 mL of buffer B and then eluted
with a linear gradient formed between 1200 mL of buffer B and
1200 mL of buffer B supplemented with 0.3 M potassium
chloride. All gradients for protein purification were formed with
Chemical structures of the product benzylic alcohols were
verified by proton NMR spectroscopy (Bruker WM-360) and IR
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Structure-Activity Studies on Sulfotransferase STa
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 69
Ta ble 1. P u r ifica tion of Su lfotr a n sfer a se STa
a standard gradient elution apparatus in which the reservoir
(containing the higher ionic strength buffer) and the mixing
chamber (initially containing the lower ionic strength buffer)
were of identical diameter and volume. The major peak of
DHEA sulfotransferase activity was eluted between 0.08 and
0.21 M potassium chloride; a smaller peak of DHEA sulfotrans-
ferase activity followed at higher salt concentrations. Phenol
sulfotransferases, as determined by assay of column fractions
with 4-nitrophenol, eluted after both peaks of DHEA sulfotrans-
ferase activity and thus were well separated from STa. The
fractions containing the major DHEA sulfotransferase activity
were pooled, concentrated to 20 mL in a stirred ultrafiltration
cell equipped with a PM10 membrane (Amicon, Inc., Beverly,
MA), and then dialyzed overnight (Spectra/Por tubing) against
two 2-L portions of buffer C (10 mM potassium phosphate (pH
6.8), 0.25 M sucrose, 3 mM 2-mercaptoethanol, and 0.05% v/v
Tween-20).
volume protein
act.a
sp act.a
purification step
(mL)
(mg)
(units) (units/mg)
100000g supernatant
DEAE-Sephadex A-50
hydroxylapatite
102
25
4.5
1.3
2530
293
1960
750
172
99
0.8
2.6
2.4
1.1
68
88
PAP-agarose
a
Enzymatic activity refers to sulfation of dehydroepiandros-
terone at pH 5.5 as described in Materials and Methods.
Following dialysis, the protein was applied to a 2.5 × 12 cm
column of hydroxylapatite that had been pre-equlibrated with
buffer C. The column was washed with 80 mL of buffer C and
then eluted with a linear gradient formed between 450 mL of
buffer C and 450 mL of buffer C containing 0.7 M potassium
phosphate (pH 6.8). One minor peak of DHEA sulfotransferase
activity was eluted between 0.1 and 0.25 M potassium phos-
phate, and the major portion of DHEA sulfotransferase activity
was eluted between 0.35 and 0.55 M potassium phosphate. No
4-nitrophenol sulfotransferase activity was detected in either
of these two DHEA sulfotransferase peaks. The major peak of
DHEA sulfotransferase activity was pooled, concentrated by
ultrafiltration to approximately 2 mL, and then dialyzed
overnight against two 2-L portions of buffer B.
F igu r e 1. Hydroxylapatite chromatography during purification
of STa. A representative elution profile is shown for chroma-
tography of STa on hydroxylapatite as described in Materials
and Methods. Fractions containing 15 mL of eluate were
collected, and sulfotransferase activity was located by assay at
pH 5.5 with dehydroepiandrosterone (DHEA). Fractions 37-
55 were pooled and further purified by chromatography on PAP-
agarose.
Hydroxysteroid sulfotransferase STa was further purified by
applying it slowly (over 30 min) to a column of PAP-agarose
(1.5 × 6.0 cm) that had been pre-equilibrated with buffer B.
After washing the column with 60 mL of buffer B, the enzyme
was eluted with a linear gradient formed between 120 mL of
buffer B and 120 mL of buffer B containing 10 µM PAP. STa
was eluted between 0.5 and 5 µM PAP. The STa-containing
fractions were pooled and concentrated by ultrafiltration to
approximately 1 mL. The concentration of PAP in the final
enzyme preparation was reduced by addition of 30 mL of buffer
B and concentrating the resulting solution to 1 mL by ultrafil-
tration. This dilution and ultrafiltration process was carried
out one additional time for further removal of PAP, after which
the purified protein was adjusted to a concentration of 1 mg/
mL and stored at -70 °C in glass vials. The final enzyme
displayed one band on SDS-PAGE after staining with Coo-
massie Blue. Identification of the homogeneous enzyme as STa
was confirmed by subjecting it to N-terminal amino acid
sequencing by automated Edman degradation using a Model
475A Applied Biosystems sequencer with an on-line Model 120
phenylthiohydantoin analyzer and a Model 900A control/data
system.
apparent Km values for PAPS in these reactions were 0.10 (
0.02 mM and 0.07 ( 0.01 mM for 4-methylbenzyl alcohol and
4-butylbenzyl alcohol, respectively. All apparent Km and vmax
values are presented as ( the standard error obtained by
nonlinear least squares curve-fitting (19) of the velocity data to
the Michaelis-Menten equation. Values for kcat were calculated
using the relative molecular mass for a subunit of STa, 33 124,
as determined from the deduced amino acid sequence (13).
Resu lts
P u r ifica t ion a n d Ch a r a ct er iza t ion of STa . The
modified purification procedure for hydroxysteroid sul-
fotransferase STa incorporated several alterations in the
methods developed by Watabe and co-workers (11, 12),
while retaining the significant improvements in stability
gained by incorporation of Tween-20 into the purification
steps (12). These changes included the insertion of
hydroxylapatite chromatography between the steps em-
ploying chromatography on DEAE-Sephadex A-50 and
PAP-agarose, elution of PAP-agarose with 10 µM PAP
instead of 20 mM ADP, and deletion of a step utilizing
Sephadex G-100. Data from a representative purification
of STa are presented in Table 1. As noted by Watabe et
al. (12), while the incorporation of Tween-20 into buffers
stabilizes the sulfotransferase, it also decreases resolu-
tion of the various isoforms of hydroxysteroid sulfotrans-
ferase during chromatography on DEAE-Sephadex A-50.
We addressed this problem by using hydroxylapatite
chromatography, as shown in Figure 1, and this provided
separation of STa (fractions 37-55) from a contaminating
isoform of hydroxysteroid sulfotransferase that was still
present after chromatography on DEAE-Sephadex A-50
in the presence of 0.05% v/v Tween-20.
Det er m in a t ion of Kin et ic Con st a n t s. Various alcohols
were evaluated as substrates for purified STa using a published
HPLC method for quantitation of PAP formed in the reaction
(18). Reaction mixtures of 0.03 mL total volume contained 0.25
M potassium phosphate buffer (pH 7), 8.3 mM 2-mercaptoet-
hanol, 0.2 mM PAPS, and various concentrations of the alcohols
in acetone (final concentration of acetone in the assay was no
more than 5% v/v). Reactions were initiated by addition of 1.0-
1.6 µg of enzyme, incubated at 37 °C for 10 min, and terminated
by addition of 0.03 mL of methanol. The concentration of PAP
formed in the reaction was determined by HPLC. Linear
standard curves relating HPLC peak areas to concentrations
of PAP were determined daily. At least six different concentra-
tions of each alcohol were assayed, and these included concen-
. The
trations both greater than and less than the apparent Km
apparent Km values for PAPS in the STa-catalyzed reactions
with various alcohols were assumed to be similar. This as-
sumption was verified by examining reactions with 4-methyl-
benzyl alcohol and 4-butylbenzyl alcohol. While the apparent
Km values for these two substrates differed by 14-fold, the
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70 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Ta ble 2. Su m m a r y of Kin etic Con sta n ts for STa -Ca ta lyzed Su lfa tion of Alcoh olsa
Chen et al.
vmax
n-alkanol
Km(app)
vmax
benzylic alcohol
Km(app)
vmax
seven-carbon alcohol
Km(app)
1-propanol
1-butanol
1-pentanol
1-hexanol
1-heptanol
1-octanol
1-nonanol
1-decanol
1-undecanol
1-dodecanol
1-tridecanol
610 ( 220
106 ( 16
19.2 ( 4.0
4.3 ( 0.8
2.2 ( 0.2
87.2 ( 4.2 benzyl alcohol
25.8 ( 3.0
96.4 ( 5.3 cyclohexylmethanol
2.0 ( 0.2
4.5 ( 0.4
101 ( 3.1
55.8 ( 2.0
39.2 ( 0.6
54.0 ( 3.3
41.8 ( 2.7
43.4 ( 1.2
80.7 ( 5.7 4-methylbenzyl alcohol 2.19 ( 0.3
74.9 ( 3.7 cis-4-methylcyclohexanol
70.5 ( 6.2 4-ethylbenzyl alcohol
69.7 ( 5.1 4-propylbenzyl alcohol 0.40 ( 0.02 74.7 ( 1.3 (R)-(-)-2-heptanol
65.4 ( 3.1 4-butylbenzyl alcohol 0.16 ( 0.01 70.3 ( 1.5 (S)-(+)-2-heptanol
0.67 ( 0.04 77.3 ( 2.0 trans-4- methylcyclohexanol 5.1 ( 0.2
4.7 ( 0.8
4.9 ( 0.8
7.2 ( 0.5
26.8 ( 2.0 30.6 ( 1.1
24.6 ( 8.2 21.4 ( 3.7
40.3 ( 29 13.9 ( 6.4
0.77 ( 0.11 55.5 ( 3.5 4-pentylbenzyl alcohol 0.12 ( 0.01 63.1 ( 1.5 4-heptanol
0.37 ( 0.04 51.0 ( 2.2 4-hexylbenzyl alcohol 0.20 ( 0.02 65.4 ( 2.3 1-methylcyclohexanol
0.31 ( 0.05 50.2 ( 3.3 4-heptylbenzyl alcohol 0.19 ( 0.02 67.4 ( 1.8 2-methyl-2-hexanol
0.26 ( 0.07 37.0 ( 4.0 4-octylbenzyl alcohol
0.33 ( 0.08 25.7 ( 2.1
0.23 ( 0.03 10.0 ( 0.4
0.29 ( 0.03 58.2 ( 2.2 3-ethyl-3-pentanol
1-tetradecanol 0.23 ( 0.08 5.7 ( 0.6
a
Values for apparent Km are expressed in mM, and values for vmax are expressed as nmol min-1 (mg of STa)-1
.
F igu r e 2. Catalytic efficiency (kcat/Km) of STa with n-alkanols.
Determination of kinetic constants for primary alcohols with
structures CnH2n+1OH (n ) 3-14) was carried out as described
in Materials and Methods.
The purified enzyme displayed a single band on SDS-
F igu r e 3. Influence of carbon chain length of primary n-
alkanols on vmax and Km values. In panel A, the maximal velocity
of the STa-catalyzed reaction is plotted as a function of carbon
chain length; the line was calculated by least squares linear
regression. In panel B, the logarithm of the Km value for each
substrate in the STa-catalyzed reaction is shown as a function
of the carbon chain length.
PAGE at a relative molecular mass of 30 200, consistent
with the previously described behavior of STa on SDS-
PAGE (11). Automated sequence analysis of the first 21
amino acids of the N-terminus of the purified protein
yielded a sequence identical to that deduced by Ogura et
al. (13) from the cDNA for sulfotransferase STa (i.e., Pro-
Asp-Tyr-Thr-Trp-Phe-Glu-Gly-Ile-Pro-Phe-Pro-Ala-Phe-
Gly-Ile-Pro-Lys-Glu-Thr-Leu-).
in the catalytic efficiency of STa upon varying the carbon
chain length of n-alkyl substituents on para-substituted
benzyl alcohols were also examined. As seen in Figure
4, an optimum value of kcat/Km was observed for 4-pen-
tylbenzyl alcohol, and this was principally due to a
minimum in the apparent Km (Table 2) for this substrate.
The apparent Km for the benzylic alcohol displaying the
maximum catalytic efficiency (4-pentylbenzyl alcohol)
was 2.6-fold lower than the apparent Km for 1-decanol,
the n-alkanol that showed optimum catalytic efficiency
in the STa-catalyzed sulfation reaction.
Role of Su bstr a te Hyd r op h obicity in th e Ca ta -
lytic Efficien cy of STa . A quantitative assessment of
hydrophobic effects in the catalytic efficiency of STa-
catalyzed sulfation of alcohols was obtained by correlat-
ing the logarithms of the kcat/Km values for several
substituted benzylic alcohols with the logarithms of the
partition coefficients for these substrates as determined
with octanol and 0.25 M potassium phosphate buffer (pH
7.0). As seen in Figure 5, the results of these experi-
ments indicated a good correlation between the hydro-
phobicity of the benzylic alcohol and its kcat/Km value as
a substrate for STa. We also examined the more general
applicability of this relationship by correlation of our
In flu en ce of Ca r bon Ch a in Len gth on th e STa -
Ca ta lyzed Su lfa tion of Alip h a tic P r im a r y Alcoh ols.
Primary aliphatic alcohols with carbon chain lengths
from 3-16 were examined for their ability to serve as
substrates for the purified hydroxysteroid sulfotrans-
ferase STa. Kinetic data for alcohols containing from
3-14 carbons are presented in Table 2; 1-pentadecanol
and 1-hexadecanol were not substrates for the STa. The
second-order rate constant, kcat/Km, for each STa-cata-
lyzed reaction is a measure of catalytic efficiency of the
enzyme with that substrate. As seen in Figure 2, the
values of kcat/Km reached a maximum for 1-nonanol,
1-decanol, and 1-undecanol. The observed decline in
catalytic efficiency after a carbon chain length of 11 was
predominantly due to a linear decline in the maximal
velocity of the reaction with increasing carbon chain
length (Figure 3A). As seen in Figure 3B, the values for
apparent Km , however, declined to a value of 0.3 mM
for 1-decanol and remained relatively constant for C11-
C14.
Effect of P a r a n -Alk yl Su bstitu en ts on th e STa -
Ca ta lyzed Su lfa tion of Ben zylic Alcoh ols. Changes
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Structure-Activity Studies on Sulfotransferase STa
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 71
F igu r e 6. Relationship between the logarithm of kcat/Km for
the STa-catalyzed reaction and the logarithm of the partition
coefficient (concentration in organic phase/concentration in
water) for n-alkanols. Kinetic constants for the enzymatic
reaction were determined as described in Materials and Meth-
ods. Symbols, organic phases, and references are as follows: b,
ethyl acetate (23); 0, butyl acetate (23); 9, octane (24); O,
dodecane (24); and 2, hexadecane (24).
F igu r e 4. Catalytic efficiency (kcat/Km) of STa with p-alkyl
substituted benzyl alcohols. Kinetic constants were obtained as
described in Materials and Methods.
F igu r e 5. Relationship between the logarithm of kcat/Km for
the STa-catalyzed reaction and the logarithm of the octanol/
buffer partition coefficient for benzylic alcohols. Kinetic con-
stants were determined as described in Materials and Methods.
Partition coefficients were determined with n-octanol and 0.25
M potassium phosphate buffer (pH 7.0), as described in Materi-
als and Methods.
F igu r e 7. Catalytic efficiency (kcat/Km) of STa with primary,
secondary, and tertiary alcohols with seven carbon atoms.
Kinetic constants were obtained as described in Materials and
Methods, and the chemical structure of each alcohol is shown
above the appropriate value of kcat/Km.
observed values for kcat/Km with literature values for
partition coefficients of aliphatic primary alcohols that
were determined with several different organic solvents
(Figure 6).
of HMBA catalyzed by STa at pH 7.0. Assay mixtures
contained concentrations of HMBA ranging from 16 to
500 µM, and analysis of reaction velocities at six concen-
trations of HMBA yielded the following kinetic con-
stants: apparent Km ) 0.12 ( 0.02 mM, vmax ) 8.0 ( 0.5
Ca ta lytic Sp ecificity of STa for P r im a r y, Secon d -
a r y, a n d Ter tia r y Alcoh ols. A series of seven-carbon
alcohols were examined in order to explore the relative
specificity of STa for primary, secondary, and tertiary
alcohols. As seen in Figure 7, the two primary alcohols
had the highest values of kcat/Km, and these were from
3-fold to 8-fold higher than for the secondary alcohols.
Catalytic efficiencies with tertiary alcohols were ap-
proximately 10-fold lower than those observed with the
secondary alcohols.
7-(H y d r o x y m e t h y l)-12-m e t h y lb e n z[a ]a n t h r a -
cen e. HMBA has been extensively studied (2, 7) both
due to its action as a carcinogen and because of the role
of sulfation in formation of an electrophilic metabolite
that covalently binds to nucleophilic sites on DNA. We
therefore determined kinetic constants for the sulfation
nmol of product formed (min)-1 (mg of STa)-1, and kcat
/
Km ) 2.2 ( 0.4 mM-1 min-1
.
Deh yd r oep ia n d r oster on e a n d Ch olester ol. DHEA
is a commonly used substrate for assay of STa activity.
Thus, we sought kinetic constants for DHEA in order to
compare these with other substrates examined in this
study. It was not possible to determine meaningful
values for Km, vmax, and kcat for the STa-catalyzed reaction
due to substrate inhibition. At pH 7.0, an optimal
reaction velocity of 62 nmol of product formed (min)-1
(mg of STa)-1 was observed at 20 µM DHEA. At
concentrations of DHEA greater than 20 µM, the velocity
of reaction decreased. While this substrate inhibition
prevented the calculation of meaningful kinetic constants
for direct comparison with other alcohols used in this
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72 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Chen et al.
study, it was clear that STa efficiently catalyzed the
sulfation of DHEA at low substrate concentrations.
Cholesterol was also examined as a possible substrate
for the purified STa at two concentrations of cholesterol
(0.05 mM and at saturation of the reaction mixture) and
at each of two pH values (5.5 and 7.0). Cholesterol was
not a substrate for STa under these conditions. Further-
more, assays for sulfation of 30 µM DHEA at pH 7.0
indicated that 0.4 mM cholesterol (a 0.4 mL final reaction
mixture contained 8 µL of a 20 mM solution of cholesterol
in acetone) did not inhibit STa (100 ( 2.2% of control
STa activity, n ) 3). These results indicated that
cholesterol was neither a substrate nor an inhibitor of
STa.
Exa m in a tion of P h en ols a s P oten tia l Su bstr a tes
for STa . Assays were carried out at two concentrations
of phenol (0.05 and 1.0 mM) and at each of two pH values
(5.5 and 7.0). The phenols that were tested under these
conditions included phenol, 4-aminophenol, 2-aminophe-
nol, 3-aminophenol, hydroquinone, 1-naphthol, 2-naph-
thol, 4-acetamidophenol, pentachlorophenol, m-cresol,
p-cresol, 4-ethylphenol, 4-propylphenol, 4-sec-butylphe-
nol, 4-octylphenol, 4-dodecylphenol, 9-phenanthrol, and
estrone. None of the phenols evaluated in this study
served as substrate for STa.
Discu ssion
A prerequisite to our examination of structure-activity
relationships with hydroxysteroid (alcohol) sulfotrans-
ferase STa was the availability of sufficient quantities
of enzyme that was both homogeneous and stable.
Watabe and co-workers (12) have described the ability
of nonionic detergents such as Tween-20 to stabilize STa
during purification and storage. While this modification
was a useful improvement over their original purification
method for the enzyme (11), potential problems in the
separation of isoforms by ion-exchange chromatography
in the presence of Tween-20 have been noted (12). We
encountered similar problems with resolution of isoforms
on DEAE-Sephadex A-50. As a result, we modified the
previous purification procedures by utilizing hydroxyla-
patite chromatography to separate the isoforms after
DEAE-Sephadex A-50. Furthermore, our use of low
concentrations of PAP for elution of STa from PAP-
agarose also enhanced the purity and yield of the enzyme.
The resulting purified isoform was unambiguously iden-
tified as STa by sequence analysis of the first 21 N-
terminal amino acids.
Correlations between values for log (kcat/Km) and values
for log P clearly indicated that the hydrophobic character
of the substrate was a significant factor influencing the
catalytic efficiency of the enzyme. These results are
consistent with previous generalizations that the hydrox-
ysteroid sulfotransferases show increasing catalytic ef-
ficiencies with increasingly hydrophobic steroids (8). As
seen by the consistency in slopes of the lines in Figures
5 and 6 (values from 0.9 to 1.1), relationships between
log (kcat/Km) and log P were similar even when log P
values were determined with different organic phases.
Although catalytic efficiency increased with log P for
smaller alcohols, there was an optimum length of the
extended carbon chain for maximum catalytic efficiency
within a series. For n-alkanols optimum values of kcat/
Km were obtained for carbon chains of C9-C11. For carbon
chains above C11, kcat/Km exhibited a steady decline, and
this decrease culminated in the lack of any detectable
F igu r e 8. Structural comparison of space-filling molecular
models for n-decanol, 4-pentylbenzyl alcohol, dehydroepiandros-
terone, and cholesterol (numbered 1-4, respectively). Molecular
graphics were computed as described in Materials and Methods.
activity with 1-pentadecanol and 1-hexadecanol. With
4-alkyl substituted benzylic alcohols, optimum values of
k
cat/Km were achieved with 4-pentylbenzyl alcohol, and
this was followed by a decline in catalytic efficiency
similar to that seen with the n-alkanols.
While these relationships demonstrated that hydro-
phobic characteristics of the substrate were essential to
the catalytic efficiency of STa, limitations arising at
longer carbon chain lengths indicated limits in the size
of substrate that could be accommodated at the active
site. As seen in Figure 8, the distance from the oxygen
of the hydroxyl group to the terminal carbon atom is
similar for 1-decanol and 4-pentylbenzyl alcohol, sub-
strates displaying optimal catalytic efficiency with STa.
Furthermore, the analogous molecular dimension in
DHEA, a physiological substrate for STa, is only slightly
smaller than that seen in either 1-decanol or 4-pentyl-
benzyl alcohol. By comparison, the fourth compound
shown in Figure 8, cholesterol, has a much greater
distance between the hydroxyl oxygen and terminal
carbon than any of the three optimal structures. Cho-
lesterol was neither a substrate nor an inhibitor for STa.
The effect of increasing carbon chain length on the vmax
of the reaction with n-alkanols, as seen in Figure 3A,
followed a linear relationship that resulted in the predic-
tion that the vmax would become zero between C15 and
C16. Indeed, both 1-pentadecanol and 1-hexadecanol
were inactive as substrates for STa. Molecular models
(not shown) indicated that extended conformations of
1-pentadecanol have approximately the same distance
from the hydroxyl oxygen to the terminal carbon as in
cholesterol.
In addition to the above relationships within the two
groups of alcohols, there was also a difference between
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Structure-Activity Studies on Sulfotransferase STa
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 73
the kcat/Km values for the optimal members of the two
series. The kcat/Km value for 4-pentylbenzyl alcohol was
over 3-fold greater than the corresponding value for
1-decanol. There is, however, little difference between
these two molecules in the distance between the hydroxyl
group and the terminal carbon (Figure 8), and a differ-
ence in partition coefficient was not sufficient to explain
a higher catalytic efficiency for the benzylic alcohol.
Indeed, theoretical calculations of log P values (23, 25)
for these compounds indicate that 4-pentylbenzyl alcohol
is somewhat less hydrophobic than 1-decanol. These
considerations indicate that additional factors that con-
tribute to binding and catalysis remain to be elucidated.
One factor that is likely to contribute to the specificity
of STa is the steric environment of the hydroxyl group
that is sulfated. We therefore examined differences
between primary, secondary, and tertiary alcohols as
substrates for STa by determining the kcat/Km values for
various seven-carbon alcohols. In general, STa exhibited
the highest kcat/Km with primary alcohols, lower catalytic
efficiency with secondary alcohols, and even smaller
values for tertiary alcohols. While a decrease in hydro-
phobicity resulting from branching of the carbon skel-
etons in these alcohols (23, 25) might play some role in
these alterations of catalytic efficiency, it does not appear
to be sufficient to fully explain the differences observed.
Further investigation of steric effects at the active site
of STa will be required to more accurately interpret these
effects on catalytic efficiency.
although it was not possible to delineate specific struc-
tural features contributing to these differences (11). In
our studies, HMBA exhibited interactions with the
enzyme that were consistent with our findings on model
compounds. As might be expected from the hydrophobic
characteristics of this molecule, the apparent Km
of 0.12
mM was near the lower limit seen with substituted
benzylic alcohols. Furthermore, the relatively low vmax
for HMBA was consistent with vmax values for both the
longer chain benzylic alcohols and the n-alkanols. Thus,
our findings have helped initiate the process of uncover-
ing those molecular parameters in hydroxyl-containing
substrates that form the basis for the specificity and
catalytic function of STa. The next step in using struc-
ture-activity studies to more accurately predict the role
of STa in sulfation of specific alcohols such as the
hydroxymethyl polycyclic aromatic hydrocarbons will
undoubtedly include further investigation of fundamental
steric interactions at the active site of the enzyme,
extension of the scope of substrates for which kcat/Km data
are available, and incorporation of theoretical and ex-
perimental data on hydrophobic and steric properties of
the individual alcohols.
Ack n ow led gm en t. This investigation was supported
by United States Public Health Service Grant CA38683
awarded by the National Cancer Institute, Department
of Health and Human Services.
In addition to hydrophobic and steric factors, the
stereochemistry of chiral alcohols is another property that
might influence the catalytic efficiency of STa. Our
results on the sulfation of C7-alcohols included one
enantiomeric pair, (R)-(-)-2-heptanol and (S)-(+)-2-hep-
tanol. While the values for apparent Km were the same,
there was a small difference in vmax for the two enanti-
omers. Thus, the catalytic efficiency for the STa-
catalyzed reaction with (R)-(-)-2-heptanol was 35%
greater than that observed with (S)-(+)-2-heptanol. Al-
though this difference represents only a small degree of
stereoselectivity, examination of other chiral alcohols will
be necessary to more fully explore the stereoselectivity
of STa.
Other examples of molecules where hydrophobic and
steric effects are clearly not the only interactions impor-
tant for the substrate specificity of STa include the
phenols. None of the phenols examined in the present
study were substrates for STa, and this was consistent
with previous reports on the specificity of homogeneous
preparations of hydroxysteroid sulfotransferases 1, 2, and
3 (8-10). It is not clear, however, whether these results
were caused by poor binding of phenols at the active site
of STa, an inability to accept a sulfuryl group from PAPS,
or both. Further investigation will be needed to deter-
mine the full range of factors responsible for the dis-
crimination of STa between phenols and aliphatic alco-
hols.
Refer en ces
(1) Watabe, T., Hakamata, Y., Hiratsuka, A., and Ogura, K. (1986)
A 7-hydroxymethyl sulfate ester as an active metabolite of the
carcinogen, 7-hydroxymethylbenz[a]anthracene. Carcinogenesis
7, 207-214.
(2) Watabe, T., Ishizuka, T., Isobe, M., and Ozawa, N. (1982) A
7-hydroxymethyl sulfate ester as an active metabolite of 7,12-
dimethylbenz[a]anthracene. Science 215, 403-405.
(3) Okuda, H., Nojima, N., Watanabe, N., and Watabe, T. (1989)
Sulphotransferase-mediated activation of the carcinogen 5-hy-
droxymethylchrysene; species and sex differences in tissue dis-
tribution of the enzyme activity and a possible participation of
hydroxysteroid sulfotransferases. Biochem. Pharmacol. 38, 3003-
3009.
(4) Surh, Y.-J ., Liem, A., Miller, E. C., and Miller, J . A. (1989)
Metabolic activation of the carcinogen 6-hydroxymethylben-
zo[a]pyrene: formation of an electrophilic sulfuric acid ester and
benzylic DNA adducts in rat liver in vivo and in reactions in vitro.
Carcinogenesis 10, 1519-1528.
(5) Surh, Y.-J ., Kwon, H., and Tannenbaum, S. R. (1993) Sulfotrans-
ferase-mediated activation of 4-hydroxy- and 3,4-dihydroxy-3,4-
dihydrocyclopenta[c,d]pyrene, major metabolites of cyclopenta-
[c,d]pyrene. Cancer Res. 53, 1017-1022.
(6) Glatt, H., Pauly, K., Pie´e-Staffa, A., Seidel, A., Hornhardt, S., and
Czich, A. (1994) Activation of promutagens by endogenous and
heterologous sulfotransferases expressed in continuous cell cul-
tures. Toxicol. Lett. 72, 13-21.
(7) Surh, Y.-J ., Liem, A., Miller, E. C., and Miller, J . A. (1991) Age-
and sex-related differences in activation of the carcinogen 7-hy-
droxymethyl-12-methylbenz[a]anthracene to an electrophilic sul-
furic acid ester metabolite in rats; possible involvement of
hydroxysteroid sulfotransferase activity. Biochem. Pharmacol. 41,
213-221.
(8) J akoby, W. B., Sekura, R. D., Lyon, E. S., Marcus, C. J ., and
Wang, J .-L. (1980) Sulfotransferases. In Enzymatic Basis of
Detoxication (J akoby, W. B., Ed.) Vol. II, pp 199-228, Academic
Press, New York.
While future refinements to the structure-activity
relationships established in the present study will further
enhance the accuracy of predictions about the specificity
and catalytic efficiency of STa, we have already begun
to explore the application of our results on model
compounds to molecules such as the carcinogen HMBA.
A previous examination of the relative rates of STa-
catalyzed sulfation of HMBA and three other (hydroxym-
ethyl)arenes at a single substrate concentration sug-
gested the influence of substrate structure on catalysis,
(9) Lyon, E. S., Marcus, C. J ., Wang, J .-L., and J akoby, W. B. (1981)
Hydroxysteroid sulfotransferase. Methods Enzymol. 77, 206-213.
(10) Lyon, E. S., and J akoby, W. B. (1980) The identity of alcohol
sulfotransferases with hydroxysteroid sulfotransferases. Arch.
Biochem. Biophys. 202, 474-481.
(11) Ogura, K., Sohtome, T., Sugiyama, A., Okuda, H., Hiratsuka, A.,
and Watabe, T. (1990) Rat liver cytosolic hydroxysteroid sul-
fotransferase (sulfotransferase a) catalyzing the formation of
reactive sulfate esters from carcinogenic polycyclic hydroxym-
ethylarenes. Mol. Pharmacol. 37, 848-854.
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+

74 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Chen et al.
(12) Watabe, T., Ogura, K., Satsukawa, M., Okuda, H., and Hiratsuka,
A. (1994) Molecular cloning and functions of rat liver hydroxys-
teroid sulfotransferases catalysing covalent binding of carcino-
genic polycyclic arylmethanols to DNA. Chem.-Biol. Interact. 92,
87-105.
(13) Ogura, K., Kajita, J ., Narihata, H., Watabe, T., Ozawa, S., Nagata,
K., Yamazoe, Y., and Kato, R. (1990) cDNA cloning of the
hydroxysteroid sulfotransferase STa sharing a strong homology
in amino acid sequence with the senescence marker protein
SMP-2 in rat livers. Biochem. Biophys. Res. Commun. 166, 1494-
1500.
(14) Ogura, K., Satsukawa, M., Okuda, H., Hiratsuka, A., and Watabe,
T. (1994) Major hydroxysteroid sulfotransferase STa in rat liver
cytosol may consist of two microheterogeneous subunits. Chem.-
Biol. Interact. 92, 129-144.
(15) Sekura, R. D. (1981) Adenosine 3′-phosphate 5′-phosphosulfate.
Methods Enzymol. 77, 413-415.
(19) Perrella, F. W. (1988) EZ-FIT: A practical curve-fitting micro-
computer program for the analysis of enzyme kinetic data on IBM-
PC compatible computers. Anal. Biochem. 174, 437-447.
(20) Nystrom, R. F., and Brown, W. G. (1947) Reduction of organic
compounds by lithium aluminum hydride. II. Carboxylic acids.
J . Am. Chem. Soc. 69, 2548-2549.
(21) Panetta, C. A., Garlick, S. M., Durst, H. D., Longo, F. R., and
Ward, J . R. (1990) Synthesis of 4-alkyl-2-iodosobenzoic acids:
potent catalysts for the hydrolysis of phosphorous esters. J . Org.
Chem. 55, 5202-5205.
(22) Bellucci, C., Gualtieri, F., and Chiarini, A. (1987) Negative
inotropic activity of para-substituted diethyl benzylphosphonates
related to fostedil. Eur. J . Med. Chem. 22, 473-477.
(23) Hansch, C., and Leo, A. (1979) Substituent Constants for Cor-
relation Analysis in Chemistry and Biology, J ohn Wiley and Sons,
New York.
(24) Aveyard, R., and Mitchell, R. W. (1969) Distribution of n-alcohols
between water and n-alkanes. Trans. Faraday Soc. 65, 2645-
2653.
(16) Nose, Y., and Lipmann, F. (1958) Separation of steroid sulfoki-
nases. J . Biol. Chem. 233, 1348-1351.
(17) Bensadoun, A., and Weinstein, D. (1976) Assay of proteins in the
presence of interfering materials. Anal. Biochem. 70, 241-250.
(18) Duffel, M. W., Binder, T. P., and Rao, S. I. (1989) Assay of purified
sulfotransferase suitable for reactions yielding unstable sulfuric
acid esters. Anal. Biochem. 183, 320-324.
(25) Leo, A. (1993) Calculating log Poct from structures. Chem. Rev.
93, 1281-1306.
TX950065T
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