Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2~18~
COLORIMETRIC DET~ INATI~N OF PEROXIDASE AND PEROXID~
Field of the Invention
The present invention rela-tes -to the colorimetric
determination of peroxidases and peroxide. In particular, the
present invention relates to -the colorimetric de-termination of
peroxidases, peroxide, compounds which can be directly or
indirectly oxidized to yield a stoichiometric quantity of
10 hydrogen peroxide, and catalys-ts which directly or indirectly
oxidize compounds to yield a stoichiometric quantity of hydrogen
peroxide.
Backqround of the Invention
The generation of hydrogen peroxide (H2O2) when
polymorphonuclear leukocy-tes and macrophages are subjected to
certain membrane stimuli, and the reaction of H2O2 with phenol
red in the presence of horseradish peroxi.dase to produce a
20 reaction product which changes the color of the system from red
to yellow, have been described [Pick, et al., Journal of
Immunoloqical Methods, Vol. 38, pages 161-170 (1980)]. The
described yellow color of such system changes to purple-mauve
when the pH is raised to 12.5 wherein the amount of H2O2 is
25 determined by measuring the absorbance oE the pu.rple-mauve
system at 610 nanometers against the absorbance of a buffered
phenol red solution. Similarly, the determination of
2~8~ ~
neutrophil-genera-tecl lipid peroxides by the oxidation of phenol
xed has also been described [Moslen, et al., Journal_of
mm oqical Methods, Vol. 98, pages 71~76 (1987)], as well as
the determination of the degree of oxidation of molten fats
5 utilizing various oxidizable dyes in a very strongly alkaline
medium (German Patent No. 2,630,052 and German Patent No.
2,5~3,543).
However, such methods require treatment with s-trong alkali
solutions of pH 10 or above, either during or after oxidation of
10 the dyes emp:Loyed therein, in order to achieve the desired
change in color.
Summary of the Invention
The present invention provides a method -to determine the
presence or amount of peroxidase, hydrogen peroxide (~I2O2), and
substances which are capable of generating hydrogen peroxide, or
catalyzing the generation of hydrogen peroxide. According to
such method, a sulfonephthalein dye or a phthalein dye having a
20 major absorbance peak in the visible region, preferab:Ly at a
wavelength from between about 500 nanometers to about 700
nanometers, is subjected to oxidative extinguishment without
strong alkali treatment.
The method is performed by forming a reaction system
25 comprising such sulfonephthalein dye or phthalein dye and:
(a) a test sample containing an unknown amoun-t of peroxidase;
and an amount of H2O2 sufficient to cause extinguishment of the
2 ~
absorbance peak of such dye whereby the amount of peroxidase can
be determined by monitoring such extinguishment; or
(b) a test sample containing an unknown amount of a compound
which is capable of undergoing a reaction wi-th oxygen to form
5 H2O2; peroxidase; and, if necessary, an amount of an enzyme or
other catalyst which causes -the compound to undergo the reaction
which forms ~22r wherein the H2O2 which is generated causes
extinguishment of the absorbance peak of such dye whereby the
amount of the compound can be determined by monitoring such
10 extinguishment; or
(c) a test sample containing an unknown amount of a peroxide,
and an amount of peroxidase, wherein the peroxide causes
extinguishment of the absorbance peak of such dye whereby -the
amount of the peroxide can be determined by monitorinq such
~5 extinguishment; or
(d) a test sample containing an unknown amount of a catalyst
which reacts with a compound in the presence of oxygen to form
H2O2, and an amount of a peroxidase, wherein the amount of H2O2
which is generated causes such extinguishment of the absorbance
20 peak of such dye whereby -the amount of the catalyst can be
determined by monitoring rate of such extinguishment.
Once extinguishmen-t of the absorbance peak of such dye has
been monitored and the value thereo-f determined, such
extinguishment value is compared with previously prepared
25 standards to determine the unknown amount. The extinguishment
of the absorbance peak can be monitored by an instrument or may
be visually monitored by observing the change of hue.
20981~9
The method of the present invention is particularly useful
for determining the presenGe or amount of peroxidase which may
be bound to, for example, an antibody or analyte, such as in an
immunoassay method or system, or for determining an analyte or
5 an enzyme or other catalyst which participates in the generation
of hydrogen peroxide by, for example, enzymatic oxidation,
wherein the amount of hydrogen peroxide so produced, or rate of
production thereof, is related to the amount of analyte or
enzyme present in a test sample. The dye employed according to
10 the present invention may be present in solution as a free form
thereof, or it may be bound, absorbed or otherwise affixed to a
wettable, solid matrix according to methods known in the art.
Brief Description of the Drawinqs
Figs. 1-3 are graphs which illustrate changes in absorbance
of a dye in a liquid phase spectrophotometric assay for a
peroxide generating analyte according to the present invention.
Figs. ~-5 are graphs which illustrate the general principal
2~ of the change in color hue of dyes employed according to the
~- present invention.
Fig. 6 illustrates the general structure of nonionized
sulfonephthalein dyes as contemplated by the present invention.
Fiy. 7 illustrates the general structure of ionized
25 sulfonephthalein dyes as contemplated by the present invention.
2~S~ ~9
Fig. ~ illus-trates the general s-tructure of nonionized
phthalein dyes as contemplated according to the presen-t
invention.
Figs. 9-13 are graphs which illustrate the absorbance of
5 reaction mixtures employed in liquid phase kinetic assays for
peroxidase according to the present invention.
Figs. 14-17 are graphs which illus-trate the effect of pH when
performing the method according to the present invention.
Description of the Invention
As used herein, and in the appended claims, the terms
H2O2 means hydrogen peroxide; "percent" and "parts' refer to
percent and parts by weight, unless otherwise indicated; g means
15 gram or grams; mg means milligram or milligrams; ~g means
microgram or micrograms; ng means nanogram or nanograms; cm
means centimeter or centimeters; mm means millimeter or
millimeters; nm means nanometer or nanometers; A means
absorbance; 1 means liter or liters; ~1 means microliter or
20 microliters; ml means milliliter or Inilliliters; mJo means mole
percent, and equals 100 times the number of moles of the
consti-tuent designated in a composition divided by the total
number of moles in the composition; V/v means percent hy volume;
M means molar and equals the number of gram moles of a so:Lute in
25 1 liter of a solution; mM means millimolar and equals the number
of millimoles of a solute in 1 liter of a solu-tion; ~M means
micromolar and equals the number of micromoles of a solute in 1
2~9~18~
liter of a solution; ~mole means micromole or micromoles and
equals the number of microgram moles of the constituent
designated; and psi means pounds per square inch pressure. All
temperatures are in C., unless otherwise indica-ted.
According to the present invention, it has been found that
certain sulfonephthalein dyes and phthalein dyes are
progressively oxidized by H2O2 in the presence of a suitable
catalyst, such as a peroxidase enzyme, and as a result of such
oxidation, undergo a succession of changes in color hue. The
lOchange of color hue is caused by the oxidative extinguishment of
the absorbance peak of sulfonephthalein dyes and phthalein dyes
at a wavelength in the visible region, preferably from between
about 500 nanometers and abou-t 700 nanome-ters. As contemplated
by the present invention, sulfonephthalein dyes include, but are
15 not intended to be limi-ted to, bromcresol green, bromcresol
purple, bromcresol blue, bromthymol blue, thymol blue, phenol
red, and the like, and phthalein dyes include, but are not
intended to be limited to, phenophthalein, and the like.
For example, bromcresol green and bromcresol purple are
20 yellow when the amount of HzOz is sufficient to cause complete
oxidation and when there is an excess of H2O2. When oxidation
is incomplete, the hue is that which results from a blending of
the yellow oxidized dye with the unoxidized dye, which is blue
in -the case of bromcresol green and purple in the case of
25 bromcresol purple. Accordingly, by carrying out a plurality of
such oxidations of differing, known amounts of bromcresol green
or of bromcresol purple and obser~ing the hue subseyuent to
2 ~
oxidation, it is possible to select a known amount of the dye
which is sufficient to react with the amount of H2O2 formed by
enzymatic oxidation of the unknown sample or, alterna-tively, to
estimate the amount of un}cnown analyte in a sample by visually
5 determining the extent of color change in a known quantity of
dye. In addition, it possi~le to make a spectropho-tometric
measurement of the extent of the color change in a known
quantity of the dye. As would be understood by one skilled in
the art, either free peroxidase or peroxidase bound to, for
10 example, an antibody or analyte, can be combined in an reaction
system with H2O2, or a source thereof, and a dye such as
described above, which is also progressively oxidi~ed by H2O2
and undergoes a succession of changes in hue. Accordingly,
since the oxidation of such dyes by the peroxidase is ca-talytic,
15 the rate at which hue changes as a result of the oxidation is a
direct function of the concentration of the peroxidase or of any
substance to which peroxidase is stoichiometrically bound.
Similarly, a peroxide or an analyte such as glucose or
cholesterol, which can undergo an enzymatic reaction which
20 produces an amount of H2O2 or other peroxide s-toichiometrically
related to the amount of the analyte, can be combined in a
reaction system with peroxidase, a dye as heretofore described,
and, in the case of, for example, glucose, cholesterol or the
like, an enzyme which causes the glucose, cholesterol or the
25 like -to react, to produce H2O2. The extent of the enzymatic
oxidation of such dye by the H2O2 and the ex-tent of the change
in hue are a measure of the amount of the analyte present in the
aqueous system.
In particular, the amount of free or bound peroxidase, or the
unknown amount of a catalyst, is determined by monitoring the
5 rate of change of hue, preferably by monitoring the rate of
change of absorbance at a given wavelength at which oxidation of
the dye extinguishes absorbance. It is to be understood that
the amount of H2O2 or the amoun-t of an H2O2-gcnerating analyte
can be determined by monitoring such rate of change either
10 visually or with an instrument. According to a preferred visual
method, a reaction system is formed, which may or may not
include solid state chemically inert components to determine
sample volume, deliver reagents, separate components or perform
other functions, comprising peroxidase r a dye according to the
15 present invention, and either a sample containing the peroxide
to be determined or a sample of the analyte compound to be
determined, and an enzyme, if required, which causes the
compound to undergo a reaction which forms HzO2. The sys-tem is
then observed to determine the hue. By comparing the resulting
20 hue with those of a standard chart of the hue resulting from
known and varying amounts of H2O2, it is possible to make a
semiquantitative visual determination, or a quantitative
determination with an instrument, of the amount of the peroxide
or of the peroxide-generating analyte under determination.
25 Although it is preferred to employ an instrument to measure
absorbance when peroxidase is being kinetically determined, a
semiquantitative visual determination of peroxide, glucose,
209~ 8~
cholesterol or the li~e substances capable of generating
peroxide, may be accomplished as well.
While a number of phthalein and sulfonephthalein dyes (Fig. 6
illustrates non~ionized sulfonephthalein dyes, Fig. 7
5 illustrates ionized sulfonephthalein dyes, and Fig. 8
illustrates non-ionized phthalein dyes) are susceptible to
oxidation by H2O2 as catalyzed by peroxidase and can be employed
according to the present invention, it is preferred that such
dyes possess the following properties:
(a) an oxidatively extinguishable light absorbance peak in
the range of 500 to 700 nm (green to violet) of sufficient
magnitude to mask a non-oxidatively bleachable peak of much
lower magnitude at ca. 390 to 450 nm (yellow to orange)
possessed by many, if not all, phthalein and sulfonephthalein
15 dyes. Phenol red, as shown in Fig. S, possesses an oxidatively
~ extinguishable absorbance peak at ca. 560 nm while a peak at ca.
; 430 nm is resistant to oxidative bleaching. However, even
before oxidation, the 430 nm (yellow-orange) peak is of
substantially greater magnitude than the 560 nm peak so that
20 peroxidase-catalyzed oxidation results in a hue change (scarlet-
; orange to orange) which can be followed instrumentally at 560 nm
or visually with some degree of difficulty; and
(b) a pKa of ionization of the phenolic hydroxyls (and,consequently, a shift from generally yellow acid pH color to
25 green to violet alkaline pH color) of less than 8, preferably
less than 7, so that the dye will be in the ionized form and
2 0 ~
susceptible to oxidation at a pH within the range where
peroxidase is active (pH 5 to 8).
When used as pH indicators, bromcresol green ionizes and
changes color at pH 3.8 -to 5.4; bromcresol purple at pH 5.2 to
5 6.8; bromthymol blue at pH 6.0 to 7.6; phenol red at pH 6.8 to
8.2; thymol blue at pH 8.0 to 9.2; and phenolphthalein at pH 8.5
to 10. Furthermore, it has been found that while phthalein and
sulfonephthalein dyes may be oxidatively bleached at any pH
abo~e the lower limit of their color shift ranges (e.g. pH 5.2
10 for bromcresol purple), tha-t the kinetics (speed) of oxidation
are greatest at a pH close to the bottom of the range of their
color shift. However, the magnitude of the oxidatively
bleachable absorbance peak a-t 500-700 nm is quite low at a pH
near the lower limit of ~hat at which the color change occurs
15 and increases rapidly with increase in pH to a maximum at and
above the upper pH limit of the color shift range. Thus,
sensitivity of assays is a compromise between maximum speed
(kinetics) at a pH near the lower limit at which the color shift
begins (phenolic ioni~ation) and maximum discrimination between
20 pre-oxidation and post-oxidation absorbance peak intensity (500-
700 nm) at a pH near or above the upper pH limit at which the
color shift occurs. It is apparent, therefore, that the pH of
the buffer in which the assay is run can be manipulated to
maximize (1) the speed of reaction (a pH toward the lower end of
25 the color shift range; desirable for many instrumental
applications or for assays for analytes expected to be present
in low concentrations), or (2) the magnitude of the difference
20~8189
between initial and final absorbance values and, thus, the
apparent extent of hue changes (a pH toward the higher end of
the color shift range, desirable for many visual
semiquantitative assays, or for assays for analytes expected to
5 be present in relatively high concentrations and thus requiring
a large dynamic range for quantitation).
For general applications, the optimal pH compromise between
speed and magnitude of color change appears to be in the upper
third of the pH-induced pre-oxidation ionization range, e.g., pH
10 7.5 to 7.8 for phenol red, pH 5.2 to 5.6 for bromcresol green,
pH 6.5 for bromcresol purple, and pH 7.4 for bromthymol blue.
For convenience, most assays described herein were conducted
either at pH 7.4 (in a 50 mM sodium phosphate buffer solution)r
or at pH 6.0 (in a 50 mM potassium 2-(N-morpholino)
15 ethanesulfonate ["MES"; buffer solution). Even though it is
recogniæed, therefore, that many reactions may not have been
performed at optimal pH conditions for assay sensitivity, the
examples given herein are sufficient to illustrate applications
of the instant invention to assays for peroxide, substances
20 capable of generating peroxide, and peroxidase enzyme activity.
It will be readily apparent to those skilled in the art that,
due to the subtractive nature of the apparent shift in hue which
occurs upon oxidation of bromcresol green and the like, i. e.,
an absorbance peak at one wavelength is extinguished, unmasking
25 another peak, rather than a true wavelength shift, the present
.inventi.on can be modified to measure different ranges of analyte
concentrations simply by altering dye concentration so as to
11
make the range between 0% and 100% oxidized dye the same order
of magnitude as the range in anticipated analyte concentrations.
The present invention ~ill now be illustrated, but is not
intended to be limited, by the following examples:
EXAMPLE 1
Liquid Phase SPectrophotometric AssaY For Glucose
A spectrophotometric assay for the kinetic determination of
10 glucose was performed employing molecular oxygen, glucose
oxidase and bromcresol purple. The glucose oxidase served as a
catalyst to oxidize glucose in solution to gluconic acid and
hydrogen peroxide wherein the hydrogen peroxide in the presence
; of the peroxidase oxidized the bromcresol purple. Such
15 oxidation extinguished absorbance by the dye at 589 nm wherein
hydrogen peroxide was quantified by measuring the extinction of
absorbance at 589 nm.
Solutions were prepared containing:
(a) 0.067 mg~ml bromcresol purple in 50 mM sodium
phosphate buffer (pH 7.4);
(b) 1 mg/ml horseradish peroxidase (Amano International
Enzyme Company) in 50 mM sodium phosphate buffer (pH 7.4);
and
(c) 10 mg/ml glucose oxidase in 50 mM sodium phosphate
buffer (pH 7.4).
Reactions were conducted by preparing mixtures at ambient
temperature (ca. 22) in 2 ml spectrophotometric cuvet-tes by
209318~
mixin~ 1.5 ml of the bromcresol purple solution, 20 ~1 of the
glucose oxidase solution, 15 ~1 of the horseradish peroxidase
solution and 0.5 ml of water or 0.5 ml of water containing
varying amounts of D-glucose, mixing rapidly, and monitoriny
5 light absorbance at 589 nm as a function of -time. The amounts
of D-glucose ranged from 0.0 to 3.~ mg (0-20 ~mole). Figs. 1,
2, and 3 are graphs which illustrate absorbance at 589 nm as a
function of time for reaction mixtures which contained,
respec-tively, 0, 0.25 and 0.5 ~mole/ml glucose. Fig. 1 shows
10 that, with no peroxide added, the absorbance at 589 nm remained
the same, slightly under 3.0, for 5 minutes. Fig. 2 shows a
decrease in absorbance, when the reaction mixture contained 0.5
~mole glucose, the slope of the curve being estimated to be
0.27. Fig. 3 shows a faster decrease in absorbance, when the
15 reaction mixture contained 1.0 ~mole ~lucose, the slope o~ the
curve being estimated to be 2.8.
The three traces shown in Figs. 1, 2 and 3, as well as others
described herein, were -taken with a Beckman DU-70 spec-tropho-
tometer (Beckman Instruments), 1.0 cm pathlength, with an
20 incandescent light source, and scanning was at a rate of 15 mn
per second.
Additional in~estigation of the reaction system of Example 1
indicated that (i~ when the system contained less than 0.4 ~mole
glucose, the rate of change of absorbance was too low fo:r the
25 techni~ue to be used for a reli.able determination of glucose,
and (ii.) when the system contained more than 1.0 ~mole glucose,
2 ~ 3
the rate of change of absorbance did not depend upon glucose
content because saturating quan-tities of H2O2 were produced.
It will be appreciated that the procedures described above in
xample 1 have not been, but could be, optimized, and that they
5 illus-trate reactions -that could be used to provide a reliable
analysis for H2O2, for glucose, and for other analytes which
react with molecular oxygen in the presence of oxidative enzymes
to produce H202. The reactions could also be used to provide a
reliable analysis for other analytes which undergo an enzymatic
10 or non-enzymatic oxidation that produces H2O2, so long as the
reaction does not involve the use of a catalyst or the like
which interferes with the oxidation of bromcresol purple or the
like by the peroxide.
For example, the procedures described above can be repeated
15 using amounts of glucose in the reaction mixture ranging from
0O4 ~mole to 1~0 ~mole to provide data for a curve showing the
slope of a curve plotting absorbance at 589 nm as a function of
glucose content. Indeed, a differentiating spec-trophotometer
which plots rate of change of absorbance at 589 nm can be used.
20 Similarly, lower concentrations of bromcresol purple can be used
to develop data where glucose in the reac-tion mixture is less
than 0.4 ~mole or higher concentrakions can be used to develop
data where glucose i5 higher than 1.0 ~mole, and like techniques
can be used to develop data to be used to analyze for peroxides
25 or for other analytes.
As will be described in greater detail hereinafter, several
procedures were carried out to demonstrate the general principal
14
2 ~ 3 ~
that the cllanc3e of hue of bromcresol green, bromcresol blue,
bromcresol purple, and -the like dyes previously discussed, as
caused by peroxidase catalyzed reaction wi-th peroxides, is -the
consequence of oxi.dative extinguishment of absorption by the
5 dyes at -the higher wavelength peaks in the li.gh-t absorption
spectra of the dyes.
In particular, the following solutions were prepared;
(a) 0.0~ mg/ml bromcresol green in 50 mM sodium phosphate
buffer (pH 6.8); and
(b) 10 mg/ml horseradish peroxidase in 50 mM sodium
phosphate buffer (pH 6.~).
A reaction mixture was prepared by mixing 1.5 ml of the
bromcresol green solution, 10 ~1 0.1 M H2O2 and, after a
spectrophotometer trace was taken, 5 ~1 of the horseradish
15 peroxidase solution was added. Two more spectropho-tometer
traces of the reaction mixture were taken, a first one taken two
minutes after the peroxidase addition, and a second one taken
four minutes -thereafter.
Fig. 4 is a graph which illustrates the three traces taken as
20 described in the preceding paragraph, 325 nm to 700 nm, wherein
curve A is the pre-oxidation trace taken before -the peroxidase
addition, curve B is the trace taken two minutes after the
peroxidase addition, and curve C is the trace taken four minutes
after the peroxidase addition. It is reaclily apparent from Fig.
25 4 that oxidation of bromcresol green progressively extinguishes
absorption a-t 612 nm (blue) while affec~inc3 neither the
magnitude nor the position of the absorption peak a-t ca. 400 nm
2~9~8~
(yellow). As this oxidation progresses, i-t is visually apparent
-that the solution is changing from blue through shades of blue-
green, green and yellow-green to, finally, a deep golden yellow.
Instrumentally, the reaction may be followed at a single
5 wavelength, the progressively diminishing peak being at 612 r~l.
Bromcresol purple and bromthymol blue, and well as other
phthalein dyes and sulfonephthalein dyes, such as described
above, when oxidized, follow patterns that are qualitatively
similar to that followed by bromcresol green, differing only in
lO the specific wavelengths at which maximum and minimum absorption
occurs, in speed of change, and in the magnitude of absorption.
For example, the major absorbance peak for bromcresol purple is
at 589 nm, while the minor absorbance peak is at 400 nm. It
will be appreciated that traces similar to those of Fig. 4 of
15 the system described in Example l which contained 1.0 ~mole
glucose would have the general appearance of Fig. 4 and from the
data of Fig. 3 that absorbance at the major absorbance peak ~at
589 nm) would be slightly less than 3.00 units before the
peroxidase addition, about 1.95 after one minute, about 0.6
20 after two minutes, about 0.3 after three minutes, and about 0.16
after five minutes.
The path followed by phenol red, when oxidized, was also
i.nvestigated by preparing solutions containing (a) 0.067 mg/ml
phenol red in 50 mM sodium phosphate buffer (pH 7.4), and (b~ 1
25 mg/ml horseradish peroxidase in 50 mM sodium phosphate buffer
(pH 7.4).
2 ~ L ~ ~
~ reaction mixture was then prepared by mixing 1.5 ml of the
phenol red solution, 50 ~l of 0.1 M H2O2 and, after a
spectrophotometer trace was taken, 15 ~l of the horseradish
peroxidase solution. Another spectrophotometer trace of the
5 reaction mixture was taken fifteen minutes after the peroxidase
addition.
Fig. 5 is a graph which illustrates the -two traces taken as
described in the preceding paragraph (400 to 700 nm) wherein
curve ~ is the pre-oxidation trace taken before -the peroxidase
10 addition, and curve B is the trace taken fifteen minutes after
the peroxidase addition. It will be seen from Fig. 5 that
phenol red follows a path similar to that followed by bromcresol
~reen, but differs in that before oxidation, the "yellow"
(actually orange) peak near 430 nm is quantitatively much larger
15 than the oxidatively rëduced "red" peak near 560 nm.
As is shown in Fig. 4, bromcresol green has two major light
absorbance bands, one at about 625 nm and one a-t about 405 nm.
Similarly, bromcresol purple has a major light absorbance bancl
at about 589 nm and another at about 400 nm. The absorbances at
20 625 nm and at 589 nm are markedly greater than those at the
wavelengths of the other major absorbance bands. When these
dyes are oxidized by H2O2 in the presence of peroxidase, the
absorbances at 625 nm and S89 nm are extinguished and, as a
consequence, the changes in hue and in absorbance are large.
25 Brom-thymol blue functions similarly with absorption maxima at
618 mn (extinguished by oxidation) and 405 nm (not
extinguished).
2 ~
EXAMPLE 2
Solid Phase Visual Assay For Hydroqen Peroxide
Filter paper discs (Whatman No. 1), 6.5 mm in diameter, were
5 impregnated with a 5 ~1 portion of a solution of 7.5 mg/ml
bromcresol green in ethanol, and dried. Each of the dried discs
were then impregnated with a 5 ~1 portion of an aqueous solution
which contained 0.08 percent of horseradish peroxidase in 60 mM
sodium phosphate buf~er (p~ 7.4), and dried. Finally, each of
10 the discs was impregnated with a 5 ~1 portion of an aqueous 0.1
N sodium nonanoate solution and dried. ~he dried discs were
placed in wells of standard 96-well matrix microtiter plates,
and a 5 ~1 portion of water or of up to 20 mM hydrogen peroxide
in water was titrated onto each disc. After the color
15 development reached equilibrium (ca. 20 minutes at room
temperature, ca. 22), the discs were dried at room temperature
and their colors were observed. There was a visually
observable, successive progression in color on the discs from
deep royal blue (O mM peroxide~ through shades of blue-green,
20 green and yellow-green (1-5 mM peroxide) to yellow (8 mM
peroxide) to orange-yellow (10-20 mM peroxide~. Similar results
were obtained when 5 ~1 portions of a solution containing 7.5
mg/ml bromcresol green in 50 mM sodium phosphate buffex (pH 7.4)
was simply pipetted into microtiter plate wells, followed by 5
25~1 of 1 mg/ml horseradish peroxidase and 5 ~1 of water or of
aqueous hydrogen peroxide, except that -the orange-yellow hue at
high peroxide concentrations was not readily observable in such
1~
2 ~
aqueous systems. The various colors formed on the discs were
(semiquantitatively) proportional to peroxide administered, and
appeared to be stable for an indefinite period of time, and did
not continue to oxidize, perceptibly, at room temperature.
A qualitatively similar, nonoptimized, assay for
cholesterol/cholesterol esters in plasma was performed in which
cholesterol/cholesterol ester~containing blood plasma was
applied to a porous polystyrene cylinder impregnated with
cholesteryl ester esterase and cholesterol oxidase (oxidizing
10 cholesterol in the presence of atmospheric oxygen to
cholestanone plus hydrogen peroxide) and placed upon similarly
treated paper discs in place of the water or hydrogen peroxide
solution. The degree of "yellowness" of the discs appeared to
be directly proportional to the amount of cholesterol applied.
It will be appreciated that there are many methods known in
the art for affixing the peroxidatively oxidizable chromogenic
dyes to solid phase matrices in such a manner as to bind them
while leaving them accessible to and reactive wi-th liquid phase
analytes and reagents as described herein. It will also be
20 appreciated that there are many methods known in the art for
deriving peroxides by oxidation, either chemical or enzymatic,
from a variety of potential analytes, and the techniques
described herein can be used to determine the amount of peroxide
formed by such procedures.
19
~98189
EXAMPLE 3
Liquid Phase_Kinetic ~ssay For Peroxidase
A liquid phase kinetic assay for peroxidase was performed by
5 first preparing the following solutions:
(a) 0.067 mg/ml bromcresol purple in 60 mM sodium phosphate
buffer ~pH 7.4);
~ b) 0-1 mg/ml horseradish peroxidase in 50 mM sodium
phosphate buffer (pH 7.4); and
(c) 100 mM H202 in water.
Reactions were conducted by preparing mixtures at ambient
temperature (ca. 22) in 2 ml spectrophotometric cuvettes by
mi~ing 1.5 ml of the bromcresol purple solution, 50 ~1 of the
H2O2 solution, and 10 ~1 of solution (b) containing from 0 to 1
15 mg/ml of the horseradish peroxidase, mixing rapidly, and then
monitoring light absorbance at 5~9 nm as a function of time.
Figs. 9, 10, 11, 12 and 13 are graphs which illustrate the
data, showing absorbance at 589 nm as a function of time for
reaction mixtures which contained 1.0, 0.5, 0.2, 0.1 and 0.05
20 mg/ml horseradish peroxidase, respectively, and include
tangential lines which represent the slopes of the straight
portions of the curves. The slopes calculated for each the
: curves are set forth in Table I below:
:'~
;,' ,, ..
,
2 ~
TABLE l
Fiqur_ Peroxidase (~q~ Slope (OD/min
9 100 2.85
1.73
ll 20 0.76
12 lO 0.32
13 5 0.15
10 It will be noted that, within the conditions and concentrations
of peroxidase tested (0-100 ~g horseradish peroxidase per 1.5 ml
reaction volume), the maximum rate of the reaction was
approximately linearly directly proportional to the amount of
the peroxidase enzyme added.
While assays for peroxides and chemical analytes susceptible
to oxidative production of peroxides (e.g. glucose, cholesterol,
xanthine, uric acid, etc.) can be performed either kinetically
or as end-point assays, the embodiment of the invention as an
assay for substances catalyzing the oxidation of said substances
20 with the consumption of peroxides (i.e., peroxidase, and
equivalents therefor) can only be performed in a kinetic format,
with the rate of the color change reaction (extin~uishment of
one.of the absorbance peaks) proportional to the peroxidase or
the like activity present, provided the concentration of dye is
25 sufficient to saturate the enzyme reaction. While the commonly
available commerclal "peroxidase" enzyme is that obtained from
horseradish roots, numerous other natural sources in plant and
2~ 9
animal tissues and microbes are knowrl, e.g., pota-toes, white
blood cells, and various bacteria, and the enzyme is likely
widespread in many other biological materials. Also, many other
protein and non-protein subs-tances, e.y., heme, exhibi-t
5 peroxidase activity though with generally lower speed and
substrate specificity than classical peroxidase enzymes.
Qualitatively similar resul-ts were obtained when the
foregoing procedure was repeated, except that phenol red was
substituted for bromcresol purple and the change in light
10 absorbance was monitored at 560 nm instead of 589 r~. The data
for phenol red are set forth in Table 2 below:
TABLE 2
-
Peroxidase (~q) SloPe (OD/min !
100 ~.27
3.05
2.23
1.36
0.45
0 5 0 07
O O
2~981~9
EXAMPLE 4
~a
The effects of pH upon the kinetics of peroxidative bleaching
5 of sulfonephthalein dyes were demonstrated by first preparing
the following solutions:
(a) 0.067 mg/ml bromcresol green in 50 mM sodium citrate
buffer (pH 5.6),
(a') 10 mg/ml horseradish peroxidase in 50 mM sodium citrate
lO buffer (pH 5.6);
(b) 0.067 mg/ml bromcresol green in 50 mM sodium phosphate
buffer (pH 6.0),
(b') 10 mg/ml horseradish peroxidase in 50 mM sodium
phosphate buffer(pH 6.0);
(c) 0.067 ms/ml bromcresol green in 50 mM sodium phosphate
buffer (pH 6.5),
; (c') lO mg/ml horseradish peroxidase in 50 mM sodium
phosphate buffer (pH 6.5); and
(d) 0.067 mg/l bromcresol green in 50 mM sodium phosphate
20 buffer (pH 7.4),
(d') 10 mg/ml horseradish peroxidase in 50 mM sodium
phosphate buffer (pH 7.4).
Four reactions were conducted b~ preparing mixtures at ambient
temperature (ca. 22) in 2 ml spectrophotometric cuvettes by
25 mixing 1.5 ml of each of the bromcresol green solutions E (a),
(b), (c) and (d)], 10 ~l 0.1 M H2O2 and 5 ~l of each of the
horseradish peroxidase solutions, [(a'), (b'), (c') and (d')}.
23
The mixtures were mixed rapidly, and the light absorbance at 612
nm as a function of time was monitored. F':igures 14, 15, 16 and
17 illustrate graphs of the data, showing absorbance at 612 nm
as a function of time for the four reaction mixtures.
Figs. 14 through 17 demonstrate the dramatic effect of pH
upon both initial absorbance values and rates of oxidative
extinguishment of the higher wavelength :Light absorbance peak of
sulfonephthalein dyes. It will be noted that at the same
concentration of dye (0.067 mg/ml), the initial absorbance at
10 612 nm is 1.2 at pH 5.6, 2.6 at pH 6.0 and 2.8 at pH 6.5 and pH
7.4. The pH-dependent color change range of bromcresol green as
a pH indicator dye is "3.8 (yellow) -to 5.4 (blue)", although it
still appears green-blue at pH 5.6 and royal blue only above pH
6. On the other hand, maximum kinetics of oxidation were
15 fastest at pH 5.6 (6-7 OD/min). At pH 6.0, the rate was 2.1
O~/min; at pH 6.5, 0.45 OD/min; and at pH 7.4, 0.25 OD/min.
This result clearly illustrates the trade-off of maximum
distinction between initial and final absorbances at higher pH
versus more rapid kinetics at lower pH as previously discussed.
20 It is apparent from the foregoing data that it is possible to
manipulate pH to optimize the conditions used in practicing the
instant invention for applica-tions in various settings and for
differe~t purposes.
It will be apparent that many modifications and variations of
25 the invention as herein set forth are possible without departing
fxom the spirit and scope thereof, and that, accordingly, such
24
limitations are imposed only as indicated in the appended
claims.
