Trace analysis of Zn(II), Be(II), and Bi(III) by enzyme-catalyzed chemiluminescence

Article abstract of DOI:10.1021/ac950681u

A novel technique for the trace analysis of metal ions Zn(II), Be(II), and Bi(III) in bulk solutions is discussed. This technique involves the generation of a chemiluminescence signal from alkaline phosphatase catalyzed hydrolysis of a phosphate derivative of 1,2-dioxetane. Zn(II) can be determined by two methods, reactivation of the alkaline phosphatase apoenzyme and inhibition of the native enzyme. Be(II) and Bi(III) can be determined quantitatively by inhibition of the native enzyme. Subppb to ppm level detection of Zn(II), Be(II), and Bi(III) has been achieved. Initial studies with mixed metals are also reported. The technique described is rapid and sensitive and can be readily applied to the microassay of heavy metal ions.

Full text of DOI:10.1021/ac950681u

Anal. Chem. 1996, 68, 216-220
Trace Analysis of Zn(II), Be(II), and Bi(III) by
Enzyme-Catalyzed Chemiluminescence
Sanjay D. Kamtekar,† Rajiv Pande,† Madhu S. Ayyagari,†,‡ Kenneth A. Marx,† David L. Kaplan,‡
Jayant Kumar,§ and Sukant Tripathy*,†
Center for Advanced Materials, Departments of Chemistry and Physics, University of Massachusetts Lowell,
Lowell, Massachusetts 01854, and Biotechnology Division, U.S. Army Natick RD&E Center, Natick, Massachusetts 01760
added amount of the metal ions. The trace metal ion content in
a solution can thus be evaluated by measuring the increase in
activity due to reactivation of the apoenzyme. However, some
metal ions are known to inhibit the activity of the native enzyme.
An example of this is alkaline phosphatase, a dimeric enzyme
which contains four Zn(II) ions, two per monomer active site.
While apoalkaline phosphatase can be reactivated with Zn(II)
ions,1-3 Be(II), Zn(II), and Bi(III) are known to be potent inhibitors
of the native alkaline phosphatase.4-7 It is thus also possible to
develop sensitive methods for the determination of Be(II),
Zn(II), and Bi(III) concentrations on the basis of their inhibitory
effects on the native enzyme.
Techniques based on optical and electrochemical methods of
signal transduction have been devised to detect Zn(II) in the
nanomolar range.7-10 In this paper we report a novel technique
for the determination of Zn(II) ions in trace levels (ppb range)
by alkaline phosphatase regeneration and in the ppb to ppm range
by enzyme inhibition. In addition to Zn(II), Be(II) and Bi(III)
also have been determined quantitatively on the basis of their
inhibitory effect on alkaline phosphatase. The technique involves
the detection of the chemiluminescence signal generated by the
action of alkaline phosphatase on 3-[spiro(4-methoxy-1,2-dioxetane-
3-2′-tricyclo[3.3.1.1]chlorodecan-4-yl)]phenyl phosphate (CSPD)
in the presence and absence of metal ions. The effects of various
cations and anions on the enzyme activity have been studied.
Because of in situ light generation in the reaction mixture,
assembly and alignment of optical components become simple.
A novel technique for the trace analysis of metal ions
Zn(II), Be(II), and Bi(III) in bulk solutions is discussed.
This technique involves the generation of a chemilumi-
nescence signal from alkaline phosphatase catalyzed
hydrolysis of a phosphate derivative of 1 ,2 -dioxetane.
Zn(II) can be determined by two methods, reactivation of
the alkaline phosphatase apoenzyme and inhibition of the
native enzyme. Be(II) and Bi(III) can be determined
quantitatively by inhibition of the native enzyme. Subppb
to ppm level detection of Zn(II), Be(II), and Bi(III) has
been achieved. Initial studies with mixed metals are also
reported. The technique described is rapid and sensitive
and can be readily applied to the microassay of heavy
metal ions.
Activation and inhibition of enzymes by inorganic species as
a means of selective and sensitive inorganic trace analysis has
been a focus of study for the last several years.1-10 Microassay
for heavy metal ions present in samples such as biofluids,
fermentation broth, river water, and waste waters provides useful
information not only in the diagnosis of disease but also in quality
control and control of environmental contamination.
Metalloenzymes like alkaline phosphatase and carbonic anhy-
drase require heavy metal ions for their catalytic activity. These
ions are generally coordinated in specific locations in the active
sites of the enzyme and function as a cofactor in the catalytic
reaction. Removing the metal ions from the enzyme with strong
chelating agents results in the formation of the corresponding
apoenzyme lacking enzyme activity. By reexposure to the metal
ion, the apoenzyme can be reversibly activated. Therefore, the
amount of the metal complexed in the active site of the enzyme
should be directly related to the enzyme activity induced by the
coordination. Thus, enzyme activity would be proportional to the
EXPERIMENTAL SECTION
Materials. Alkaline phosphatase (3 units/ mg) from bovine
calf intestine (EC 3.1.3.1), Tris-HCl, ammonium sulfate (enzyme
grade), zinc sulfate, and p-nitrophenyl phosphate were supplied
by Sigma (St. Louis, MO) and sodium acetate, sodium chloride,
hydrochloric acid (metal free), and magnesium chloride were
purchased from Fisher Scientific (Fair Lawn, NJ). Beryllium
sulfate, bismuth nitrate, sodium fluoride, and acetylacetone were
purchased from Aldrich Chemical Co. (Milwaukee, WI). Diethyl-
amine (DEA) and CSPD were supplied as a part of a Southern-
Light Chemiluminescent Detection System by Tropix, Inc. (Bed-
ford, MA). CSPD was supplied as a 25 mM aqueous solution.
Sapphire, a luminescent amplifying material (enhancer), was also
supplied by Tropix, Inc. Deionized and distilled water was used
in all preparations. All chemicals were of maximum purity
available and were used as received.
† Department of Chemistry, University of Massachusetts Lowell.
‡ U.S. Army Natick RD&E Center.
§ Department of Physics, University of Massachusetts Lowell.
(1) Applebury, M. N.; Coleman, J. E. J. Biol. Chem. 1 9 6 9 , 244, 308-318.
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361-367.
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123, 195-200.
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274.
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199-201.
Methods. Apoenzyme was prepared by dialyzing 15-20 mL
of alkaline phosphatase (10 mg/ mL) against 2 L of 2 M ammonium
sulfate, at pH 9.0. The dialysis was carried out at 4 °C for 24 h
216 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996
0003-2700/96/0368-0216$12.00/0 © 1995 American Chemical Society

with two changes of dialysate. Ammonium sulfate was removed
by dialysis against 2 L of 0.01 M Tris-HCl, 0.01 M sodium acetate,
and 0.1 M sodium chloride at pH 9.0 with two changes of
dialysate.11 Enzyme activity was measured by following the
hydrolysis of p-nitrophenyl phosphate in 0.2 M Tris-HCl, pH 8.0,
buffer. The initial rate of formation of p-nitrophenol was recorded
at 405 nm using a Perkin-Elmer Lambda 9 UV-vis-near-IR
spectrophotometer. Zinc ion concentration in the enzyme and
apoenzyme was checked by a direct current plasma (DCP)
spectrometry (Model SMI III Spectrametrics Inc.).12 For recon-
stitution of the apoenzyme, the dialyzed enzyme was incubated
with different concentrations of Zn(II) for 2 min. Stock solutions
of Zn(II) and other metals were prepared by dissolving the spectral
grade salts in deionized distilled water. Further dilutions were
made in DEA.
Figure 1. Representation of alkaline phosphatase catalyzed hy-
drolysis of CSPD.
The assay buffer for chemiluminescence experiments was
prepared weekly as recommended by the supplier, by dissolving
DEA and MgCl2 in deionized distilled water to final concentrations
of 0.1 and 1.0 mM, respectively. The pH was adjusted to 10.0 by
adding 0.1 M HCl (metal free). Stock substrate was prepared as
a 10% solution of enhancer in DEA buffer. To 10 mL of this
solution was added 2 µL of 25 mM CSPD solution. The stock
solutions, stored at 4 °C, were brought to room temperature prior
to the reaction. The reaction mixture was prepared in a glass
test tube by first adding predetermined volumes of enzyme of
known concentration and metal ion solution to buffer. The
reaction was initiated by adding 5.0 mL of the substrate solution.
The composition of the final reaction mixture was determined after
extensive experimentation to minimize the amounts of enzyme
and CSPD and to maximize the signal-to-noise ratio.
Determination of Zn(II) by Regeneration of Activity. A typical
reaction mixture of 1.60 mL (for control) was prepared by mixing
0.5 mL of 0.4 mM CSPD solution, 1.05 mL of buffer, and 50 µL of
enzyme solution (10 mg/ mL). The reaction was initiated by
adding 0.5 mL of 0.4 mM CSPD. Zn(II) determination was done
by incubating 50 µL of apoenzyme with 50 µL of metal ion solution
for 2 min, to which 1.0 mL of buffer and 0.5 mL of 0.4 mM CSPD
were added.
Determination of Zn(II), Be(II), and Bi(III) by Inhibition of
Activity. The reaction mixture of 0.350 mL was prepared by
mixing 0.125 mL of 5.0 µM CSPD solution, 0.125 mL of metal
solution, and 0.1 mL of enzyme solution (1 mg/ mL, activity 3
units/ mL). For the control, 0.125 mL of assay buffer was added
instead of metal solution. Metal ion concentration was varied
either by adding a fixed volume of diluted metal solution or by
adding different volumes of the stock metal solution and making
up the reaction mixture volume to 0.350 mL with buffer.
Determination of Zn(II) in the Presence of Be(II) by Selective
Masking. The reaction mixture of 0.7 mL was prepared by mixing
0.25 mL of 5.0 µM CSPD solution, 0.125 mL of metal ion solution,
0.125 mL of masking agent [acetylacetone 0.02% v/ v in 0.1 M NaF
for determination of Zn(II)], and 0.2 mL of enzyme solution (at 3
units/ mL). The blanks/ control references were obtained by
replacing the metal ion solution or masking agent by assay buffer.
A photomultiplier tube (Hamamatsu, Model R 943-02; photo-
cathode material, GaAs(Cs); window, fused silica; wavelength
range, 160-930 nm), an amplifier, a photon counter, and a
microcomputer are used to collect and process the data. The
experimental setup was described in greater detail earlier.13 All
the reactions were carried out in bulk. The same test tube was
used in a given set of reactions to eliminate the effect of tube
geometry on the detected signal intensity. Less than 5 s elapsed
between the initiation of the reaction and the start of data
acquisition. The signal reached peak value in approximately 60-
70 s. Initial slopes (slope of rate of product formation or slope of
chemiluminescent photon detection) were calculated based on the
first 20 data points collected in as many seconds.
RESULTS AND DISCUSSION
Typical chemiluminescence reactions for compounds such as
lucigenin and luminol are characterized by the presence of an
oxidant, a catalyst and alkali in the reaction mixture.14 However,
1,2-dioxetanes such as CSPD release light in the absence of any
oxidant. These compounds contain a powerful oxidizing potential
built into the molecules with the presence of the inherently weak
O-O bonds in highly strained four-membered rings.15 Decom-
position of dephosphorylated CSPD is a highly exothermic
process, releasing enough energy to allow the excitation of
products to the higher electronic emissive states.16,17 Figure 1
illustrates the hydrolysis of CSPD catalyzed by alkaline phos-
phatase and subsequent photon emission. A kinetic expression
presented elsewhere was derived for the rate of generation of
chemiluminescence or from an experimental point of view, the
rate of photon detection.18 Typical postexperimental data process-
ing involved conversion of a real-time curve of photon count rate
versus time to a counts versus time curve by data integration.
The slope of the curve in its linear portion was then computed
and equated to the initial velocity of the reaction. The velocity of
the reaction is the rate of chemiluminescence signal generation.
For simplicity we measure the slope of the linear portion of the
photon count per second versus time curve as a measure of
enzyme activity. Maximum alkaline phosphatase activity for the
substrate CSPD is observed at pH 10, similar to p-nitrophenyl
(13) Ayyagari, M. S.; Kamtekar, S. D.; Pande, R.; Marx, K. A.; Kumar, J.; Kaplan,
D. L.; Tripathy, S. K. Proceedings of the 2nd International Conference on
Intelligent Materials; Technomic Publishing Co. Inc.: Lancaster, U.K., 1994;
Vol. 2, p 85.
(14) White, E. H.; Miano, J. D.; Watkins, C. J.; Breaux, E. J. Angew. Chem. 1 9 7 4 ,
86, 292-305.
(15) McCapra, F. J. Chem. Soc., Chem. Commun. 1 9 7 7 , 946-948.
(16) Turro, N. J.; Lechten, P. J. Am. Chem. Soc. 1 9 7 3 , 95, 264-266.
(17) Adam, W.; Cilento, G. Angew. Chem. 1 9 8 3 , 95, 525-538.
(18) Ayyagari, M. S.; Kamtekar, S. D.; Pande, R.; Marx, K. A.; Kumar, J.; Tripathy,
S. K.; Akkara, J.; Kaplan, D. L. Biotechnol. Prog., in press.
(11) Gettins, P.; Colman, J. E. J. Biol. Chem. 1 9 8 2 , 257, 396-407.
(12) Ingle, J. D., Jr.; Crouch, S. R. Spectrochemical Analysis; Prentice-Hall, Inc.:
Englewood Cliffs, NJ, 1988; Chapter 8.
Analytical Chemistry, Vol. 68, No. 1, January 1, 1996 217

Figure 3. Activity regeneration in apoalkaline phosphatase as a
function of solution Zn(II) concentration.
Table 1. Dependence of Reactivation of the
Apoalkaline Phosphatase Enzyme Activity on Zn(II)
Dynamic Range
enzyme activity
(units/ mL)
dynamic range
(ppb)
22
30
293
0.5-5
2-15
10-50
concentrations of Zn(II) ions. Data collection was continued for
about 1 min, well past the linear portion of the counts per second
versus time curve. In practice, the data points required to
compute slopes could be collected within 20-25 s. The initial
slopes were determined with data points collected during the first
20 s. DEA buffer in the reaction mixture plays a significant role
of scavenging the inorganic phosphate, a potential enzyme
inhibitor generated during the reaction. Inhibition rather than
enhancement was observed when similar reactions were carried
out without excess assay buffer solution. Figure 2b illustrates a
set of chemiluminescence curves for undialyzed, dialyzed, and
regenerated (by Zn(II)) enzyme. Figure 3 shows the calibration
curve for Zn(II) ion. The error bars in the figures signify the
standard deviation of five replicate measurements. Detection limit
is the concentration of metal ions determined for three standard
deviations above the mean of five replicate measurements of the
zero standard (control, without any metal ion). A detection limit
of 0.5 ppb was achieved with the chemiluminescence technique,
which is an improvement over the earlier well-established colo-
rimetric methods.3 The upper limit (maximum concentration
detected) of the dynamic range for the Zn(II) is set by the amount
of zinc present in the undialyzed enzyme (source), the concentra-
tion of the enzyme used for analysis, and the efficiency of dialysis.
Table 1 gives the dependence of the dynamic range on the protein
concentration. As in the case of native enzyme, even apoenzyme
regeneration is influenced by the presence of various metal ions.
Table 2 depicts the influence of some known interfering metal
ions on apoenzyme regeneration. The order of enhancement by
these metal ions is Zn > Co > Cd > Ca > Mg > Ba.
Figure 2. (a, top) Spectrophotometric response (405 nm) of the
hydrolysis of p-nitrophenyl phosphate to 2.5 ppb of Zn(II): (a)
undialyzed enzyme, (b) dialyzed enzyme, and (c) regenerated alkaline
phosphatase. (b) Chemiluminescence curves for undialyzed, dialyzed,
and regenerated alkaline phosphatase with 0.8 and 5.0 ppb Zn(II)
ions.
phosphate (pNPP; pH 9.8).2 Therefore, all the experiments were
carried out at room temperature and pH 10, where the enzyme
shows maximum activity. The enzyme activity can be expressed
in terms of chemiluminescence signal strength as peak photon
counts or initial slopes of counts per second versus time plot.
1 . Zn(II) Detection by Signal Enhancement. Apoalkaline
phosphatase exhibits a reduced catalytic effect on the hydrolysis
of the substrate pNPP compared to the zinc-containing native
enzyme.3 This principle has been used to develop different
techniques for the determination of zinc.6,7 Figure 2a illustrates
a typical spectrophotometric response of the hydrolysis of the
substrate pNPP by undialyzed, dialyzed, and regenerated [by
Zn(II)] enzyme, monitored at 405 nm. After adding Zn(II) ions,
the apoalkaline phosphatase regains its activity, resulting in an
increased rate of hydrolysis of pNPP. It is also evident from the
figure that dialyzing the enzyme with (NH4)2SO4 resulted in only
50% apoenzyme formation.
2 . Detection of Zinc, Beryllium, and Bismuth by Inhibi-
tion. A sample for metal ion detection often contains a variety of
inorganic and organic ionic species in various concentrations.
Therefore, different metal cations and anions were tested for their
inhibitory effect on enzyme-catalyzed chemiluminescence; Be(II),
We observed a similar effect on the chemiluminescence
generation rate from the dephosphorylation of CSPD. The
reactions were carried out as described earlier with undialyzed
and dialyzed enzyme and dialyzed enzyme incubated with different
218 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996

Table 2. Extent of Activity Regeneration in
Apoalkaline Phosphatase by Different Divalent Metal
Ions (at 10 ppb Concentration)
Table 4. Comparison of the Detection Limits for
Different Metal Ions by Various Detection Techniques
detection limit (ppm)
element
relative activity
1.0000
technique
Zn(II)
Be(II)
0.001
Bi(III)
1.8
dialyzed enzyme
currenta
0.17
0.0005
currentb
Cd2+
Zn2+
Co2+
Ba2+
Mg2+
Ca2+
1.1114 ( 0.0014
1.2920 ( 0.0016
1.2515 ( 0.0003
1.0093 ( 0.0002
1.0198 ( 0.0011
1.0329 ( 0.0001
fluorescence5
absorbancea4
electrochemical8
chemiluminescence10
absorbanceb2
0.01
0.018
1.0
0.6
0.13
0.001
0.006
a
Inhibition of native enzyme. b Activation of apoenzyme.
Table 3. Inhibitory Effect of Various Metal Ions and
Anions (at 200 ppm Concentration) on the
Enzyme-Catalyzed Chemiluminescence Signala
Table 5. Determination of Zn(II) in the Presence of
Be(II) by Selective Masking of Be(II) Ions Using
Acetylacetone and Sodium Fluoridea
cations
relative activity
anions
relative activity
Na+
1.0000
Cl-
1.0000
Be(II) (ppb)
Zn(II) (ppm)
relative activity
Al3+
0.7543 ( 0.0326
0.8245 ( 0.0486
0.7319 ( 0.0047
0.7407 ( 0.0086
0.5884 ( 0.0123
0.7649 ( 0.0075
0.9450 ( 0.0234
0.9612 ( 0.0203
0.0260 ( 0.0013
0.5154 ( 0.0170
0.3384 ( 0.0077
0.8417 ( 0.0916
0.8617 ( 0.0179
F-
1.0248 ( 0.0002
0.7256 ( 0.0215
0.9753 ( 0.0025
0.9322 ( 0.0088
0.7120 ( 0.0020
0.4705 ( 0.0035
0.9507 ( 0.0001
Ca2+
Ba2+
Pb2+
Cu2+
Mg2+
Co2+
Ni2+
Be2+
Bi3+
EDTA
SO3
CO3
NO3
0.0
0.0
0.0
0.0
0.0
0.0
8.928
8.928
13.392
13.392
17.857
17.857
1.0b
-
1.165 ( 0.016
0.279 ( 0.014b
0.916 ( 0.028
0.732 ( 0.009
0.719 ( 0.008
0.677 ( 0.018
0.663 ( 0.013
0.578 ( 0.004
0.571 ( 0.019
-
8.928
8.928
0.0
8.928
0.0
8.928
0.0
8.928
-
-
PO4
CH3COO-
Zn2+
Fe2+
Cd2+
a
Masking agent, 0.0003 M acetylacetone and 0.0178 M NaF
(acetylacetone 0.02% v/ v in 0.1 M NaF used in reaction mixture).
Enzyme activity in reaction mixture is 0.857 unit. CSPD concentration
in reaction mixture is 1.785 µM. b No masking agent used in these
sets of experiments.
a
Enzyme activity measured relative to the effect of 200 ppm NaCl.
ions. The detection limits achieved by inhibition for Zn(II),
Be(II), and Bi(III) were 0.170, 0.001, and 1.8 ppm, respectively.
These detection limits are quite comparable and, in the case of
Be, superior to those reported earlier by other techniques as
shown in Table 4.
In the above case, the effective CSPD concentration in the
reaction mixture was 1.785 µM, whereas in our earlier experi-
ments, we used a CSPD concentration of 2.631 µM with the same
enzyme concentration; the detection limits achieved by inhibition
for Zn(II), Be(II), and Bi(III) were 0.360, 0.265, and 2.6 ppm,
respectively. Hence, we believe that by lowering the enzyme and
CSPD concentrations in the reaction mixture the detection limits
can be lowered further for metal ion detection.
3 . Selective Determination of Metal Ions Using Masking
Agents. The determination of metal ions by inhibition of the
enzyme can be made metal ion specific using different masking
agents and by suitable sample pretreatment.4,19 Table 5 illustrates
the determination of Zn(II) in the presence of the potent inhibitor
Be(II) by selective masking of Be(II) using acetylacetone and
sodium fluoride. Acetylacetone effectively masks Ag, Ni, Co, and
Cu ions along with Be, and NaF masks the effect of Al, Ca, Sr,
and Mn if these interfering metal ions are present in the sample.4,19
In these experiments, different controls were performed with and
without masking agents and with either or both of the metal ions
present. It is quite evident from Table 5 that the inhibitory effects
of Zn(II) in the presence and absence of Be(II) are the same due
Figure 4. Inhibitory effect of (a) Zn(II), (b) Be(II), and (c) Bi(III) on
chemiluminescence signal.
Zn(II), and Bi(III) had the largest inhibitory effect among metal
ions evaluated along with phosphate anion (Table 3). The
dependence of enzyme activity on these metal ions as a function
of concentration is given in Figure 4. The experiments were done
at a fixed concentration of the enzyme and CSPD. The metal ion
concentration was varied to successively lower concentrations until
the difference between the initial slope of the control (absence of
any metal ion inhibitor) and one in the presence of metal ion was
negligible (within three standard deviations of the control). From
Table 3 and Figure 4, it is evident that the extent of inhibition is
in the order Be(II) > Zn (II) > Bi(III). From the data for Zn(II),
Be(II), and Bi(III), it is established that the chemiluminescence
technique could be used to detect ppb to ppm levels of these metal
(19) Pribil, R. In Applied Complexometry; Chalmers, R. A., Ed.; Pergamon Press
Ltd.: Headington Hill Hall, Oxford, England, 1982; Chapter 3, pp 64-69.
Analytical Chemistry, Vol. 68, No. 1, January 1, 1996 219

to the masking effect of the agent. Moreover, masking agent
alone had some enhancement effect on relative activity which
could be attributed to the presence of fluoride as seen before in
Table 5.
Similarly, in the selective determination of Be(II) masking
agents like thioglycolic acid, unithiol (2,3-dimercaptopropane-1-
sulfonic acid), and diethyldithiocarbamate can be used to masks
the interference from the metal ions like Zn, Hg, As, Cd, Pb, Bi,
Sb, Cu, and Ag.19
inhibition method. Zn(II) was also detected at 0.5 ppb by the
apoenzyme regeneration method. Selective determination of
Zn(II) in the presence of Be(II) was demonstrated by masking
the interfering ions with acetylacetone and sodium fluoride. Other
metal ions can be selectively determined by the proper choice of
masking agents to suppress the interfering ions.
ACKNOWLEDGMENT
The authors acknowledge support from ARO Grant DAAL03-
91-G-0064 and a joint USDA/ U.S. Army (Contract DAAK60-93-K-
0012), and we thank Dr. David Ryan and Dr. K. Shridhara Alva
for their assistance in the DCP, UV-visible experiments.
CONCLUSIONS
A novel technique for the determination of trace level of
Zn(II), Be(II), and Bi(III) is described. Alkaline phosphatase
catalyzed chemiluminescence is used to establish calibration
curves for these metal ions. In addition to rapid and sensitive
detection, the technique uses a simplified optical experimental
setup. A 0.17 ppm, 1 ppb, and 1.8 ppm level of detection was
achieved for Zn(II), Be(II), and Bi(III), respectively, by the enzyme
Received for review July 10, 1995. Accepted October 12,
1995.X
AC950681U
X Abstract published in Advance ACS Abstracts, November 15, 1995.
220 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996