WO1993012253A1

WO1993012253A1 - Colorimetric determination of peroxidase and peroxide

Info

Publication number: WO1993012253A1
Application number: PCT/US1991/009204
Authority: WO
Inventors: Steven Dale Salt; Neal Arthur Siegel
Original assignee: Abbott Laboratories
Priority date: 1991-12-09
Filing date: 1991-12-09
Publication date: 1993-06-24
Also published as: EP0618980A1; JPH07501202A

Abstract

A method to determine the presence or amount of peroxidase or hydrogen peroxide employing a sulfonephthalein dye or a phthalein dye having a major absorbance peak in the visible region, preferably at a wavelength from between about 500 nanometers to about 700 nanometers, and which absorbance peak is subject to a degree of oxidative extinguishment without strong alkali treatment, is disclosed. The method is performed by forming a reaction system comprising such sulfonephthalein dye or phthalein dye, and a test sample containing an unknown amount of (i) peroxide, (ii) a peroxidase, (iii) a compound which is capable of being oxidized to generate hydrogen peroxide, or (iv) a catalyst which participates in such oxidation, wherein the hydrogen peroxide causes peroxidase-catalyzed extinguishment of the absorbance peak of such dye whereby the unknown amount under determination can be determined by monitoring such extinguishment.

Description

COLORIMETRIC DETERMINATION OF PEROXIDASE AND PEROXIDE

Field of the Invention

The present invention relates to the colorimetric determination of peroxidases and peroxide. In particular, the present invention relates to the colorimetric determination of peroxidases, peroxide, compounds which can be directly or indirectly oxidized to yield a stoichiometric quantity of hydrogen peroxide, and catalysts which directly or indirectly oxidize compounds to yield a stoichiometric quantity of hydrogen peroxide.

Background of the Invention

The generation of hydrogen peroxide (HO ) when poly orphonuclear leukocytes and macrophages are subjected to certain membrane stimuli, ' and the reaction of H202 with phenol red in the presence of horseradish peroxidase to produce a 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 H_,0₂ is determined by measuring the absorbance of the purple-mauve system at 610 nanometers against the absorbance of a buffered phenol red solution. Similarly, the determination of neutrophil-generated lipid peroxides by the oxidation of phenol red has also been described [Moslen, et al., Journal of Immunoloqical Methods- Vol. 98, pages 71-76 (1987)], as well as the determination of the degree of oxidation of molten fats utilizing various oxidizable dyes in a very strongly alkaline medium (German Patent No. 2,630,052 and German Patent No. 2,543,543).

However, such methods require treatment with strong alkali solutions of pH 10 or above, either during or after oxidation of the dyes employed 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 (HO ), 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 major absorbance peak in the visible region, preferably at a wavelength from between about 500 nanometers to about 700 nanometers, is subjected to oxidative extinguishment without strong alkali treatmen .

The method is performed by forming a reaction system comprising such sulfonephthalein dye or phthalein dye and: (a) a test sample containing an unknown amount of peroxidase; and an amount of H₂0₂ sufficient to cause extinguishment of the 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 with oxygen to form H₂0₂; peroxidase; and, if necessary,^' an amount of an enzyme or other catalyst which causes the compound to undergo the reaction which forms HO , wherein the HO which is generated causes extinguishment of the absorbance peak of such dye whereby the amount of the compound can be determined by monitoring such 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 monitoring such 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 HO , and an amount of a peroxidase, wherein the amount of HO which is generated causes such extinguishment of the absorbance peak of such dye whereby the amount of the catalyst can be determined by monitoring rate of such extinguishment.

Once extinguishment of the absorbance peak of such dye has been monitored and the value thereof determined, such extinguishment value is compared with previously prepared 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.

The method of the present invention is particularly useful for determining the presence 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 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 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 Drawings

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. 4-5 are graphs which illustrate the general principal 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. Fig. 7 illustrates the general structure of ionized sulfonephthalein dyes as contemplated by the present invention.

Fig. 8 illustrates the general structure of nonionized phthalein dyes as contemplated according to the present invention.

Figs. 9-13 are graphs which illustrate the absorbance of reaction mixtures employed in liquid phase kinetic assays for peroxidase according to the present invention. Figs. 14-17 are graphs which illustrate 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 H 0₂ means hydrogen peroxide; "percent" and "parts" refer to percent and parts by weight, unless otherwise indicated; g means gram or grams; mg means milligram or milligrams; μg 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; μl means microliter or microliters; ml means milliliter or milliliters; means mole percent, and equals 100 times the number of moles of the constituent designated in a composition divided by the total number of moles in the composition; V means percent by volume; M means molar and equals the number of gram moles of a solute in 1 liter of a solution; mM means illimolar and equals the number of millimoles of a solute in 1 liter of a solution; μK means micromolar and equals the number of micromoles of a solute in 1 liter of a solution; /imole means micromole or micromoles and equals the number of microgram mole^'s of the constituent designated; and psi means pounds per square inch pressure. All temperatures are in °C, unless otherwise indicated.

According to the present invention, it has been found that certain sulfonephthalein dyes and phthalein dyes are progressively oxidized by HO 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 change 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 about 700 nanometers. As contemplated by the present invention, sulfonephthalein dyes include, but are not intended to be limited 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 yellow when the amount of H₂0₂ is sufficient to cause complete oxidation and when there is an excess of HO . 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 bromcresol purple. Accordingly, by carrying out a plurality of such oxidations of differing, known amounts of bromcresol green or of bromcresol purple and observing the hue subsequent^'to oxidation, it is possible to select a known amount of the dye which is sufficient to react with the amount of HO formed by enzymatic oxidation of the unknown sample or, alternatively, to estimate the amount of unknown analyte in a sample by visually determining the extent of color change in a known quantity of dye. In addition, it possible to make a spectrophotometric 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 example, an antibody or analyte, can be combined in an reaction system with H₂0 , or a source thereof, and a dye such as described above, which is also progressively oxidized by H₂0₂ and undergoes a succession of changes in hue. Accordingly, since the oxidation of such dyes by the peroxidase is catalytic, 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 an Jyte such as glucose or cholesterol, which can undergo an enzymatic reaction which produces an amount of H₂0₂ or other peroxide stoichiometrically 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 like to react, to produce H0. The extent of the enzymatic oxidation of such dye by the HO and the extent 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 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 H_,0₂ or the amount of an H₂0₂-generating analyte can be determined by monitoring such rate of change either 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, a dye according to the 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 H₂0₂. The system is then observed to determine the hue. By comparing the resulting hue with those of a standard chart of the hue resulting from known and varying amounts of H₂0₂, 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

SUBSTI determination. Although it is preferred to employ an instrument to measure absorbance when peroxidase is being kinetically determined, a semiquantitative visual determination of peroxide, glucose, cholesterol or the like 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 illustrates ionized sulfonephthalein dyes, and Fig. 8 illustrates non-ionized phthalein dyes) are susceptible to oxidation by H₂0₂ 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 dyes. Phenol red, as shown in Fig. 5, 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 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

SUBSTITUTESHEET (b) a p a of ionization of the phenolic hydroxyls (and, consequently, a shift from generally yellow acid pH color to 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 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 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 above the lower limit of their color shift ranges (e.g. pH 5.2 for bromcresol purple), that 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 at 500-700 nm is quite low at a pH near the lower limit of that at which the color change occurs 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 ionization) and maximum discrimination between 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 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 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 se iquantitative assays, or for assays for analytes expected to 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 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), or at pH 6.0 (in a 50 mM potassium 2-(N-morpholino) ethanesulfonate ["MES"] buffer solution). Even though it is recognized, 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 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 another peak, rather than a true wavelength shift, the present invention can be modified to measure different ranges of analyte concentrations simply by altering dye concentration so as to make the range between 0% and 100% oxidized dye the same order of magnitude as the range in anticipated analyte concentrations. The present invention will 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 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 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 5 phosphate buffer (pH 7.4).

Reactions were conducted by preparing mixtures at ambient temperature (ca. 22°) in 2 ml spectrophotometric cuvettes by mixing 1.5 ml of the bromcresol purple solution, 20 μl of the glucose oxidase solution, 15 μl of the horseradish 0 peroxidase solution and 0.5 ml of water or 0.5 ml of water containing varying amounts of D-glucose, mixing rapidly, and monitoring light absorbance at 589 nm as a function of time. The amounts of D-glucose ranged from 0.0 to 3.6 mg (0-20 /-mole) . Figs. 1, 2, and 3 are graphs which 5 illustrate absorbance at 589 nm as a function of time for reaction mixtures which contained, respectively, 0, 0.25 and 0.5 imole/ml glucose. Fig. 1 shows 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 0 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 reaction mixture contained 1.0 /anole glucose, the slope of the curve being estimated to be 2.8. 5 The three traces shown in Figs. 1, 2 and 3, as well as others described herein, were taken with a Beckman DU- 70 spectropho-tometer (Beckman Instruments), 1.0 cm pathlength, with an incandescent light source, and scanning was at a rate of 15 nm per second. Additional investigation of the reaction system of Example 1 indicated that (i) when the system contained less than 0.4 /αnole glucose, the rate of change of absorbance was too low for the technique to be used for a reliable determination of glucose, and (ii) when the system^' contained more than 1.0 /tmole glucose, the rate of change of absorbance did not depend upon glucose content because saturating quantities of H₂0₂ were produced.

It will be appreciated that the procedures described above in Example 1 have not been, but could be, optimized, and that they illustrate reactions that could be used to provide a reliable analysis for H₂0₂, 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 or non- enzymatic oxidation that produces H₂0₂, 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 using amounts of glucose in the reaction mixture ranging from 0.4 /"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 spectrophotometer which plots rate of change of absorbance at 589 nm can be used. Similarly, lower concentrations of bromcresol purple can be used to develop data where glucose in the reaction mixture is less than 0.4 /oriole or higher concentrations can be used to develop data where glucose is higher than 1.0 /jmole, and like techniques can be used to develop data to be used to analyze for peroxides or for other analytes. As will be described in greater detail hereinafter, several procedures were carried out to demonstrate the general principal that the change of hue of bromcresol green, bromcresol blue, bromcresol purple, and the like dyes previously discussed, as caused by peroxidase- catalyzed reaction with peroxides, is the consequence of oxidative extinguishment of absorption by the dyes at the higher wavelength peaks in the light absorption spectra of the dyes.

In particular, the following solutions were prepared; (a) 0.04 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 6.8). A reaction mixture was prepared by mixing 1.5 ml of the bromcresol green solution, 10 μl 0.1 M H₂0₂ and, after a spectrophotometer trace was taken, 5 μl of the horseradish peroxidase solution was added. Two more spectrophotometer 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 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 readily apparent from Fig. 4 that oxidation of bromcresol green progressively extinguishes absorption at 612 nm (blue) while affecting neither the magnitude nor the position of the absorption peak at ca. 400 nm (yellow) . As this oxidation progresses, it 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 wavelength, the progressively diminishing peak being at 612 nm.

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 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 the system described in Example 1 which contained 1.0 /zmole 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_f about 0.6 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 investigated by preparing solutions containing (a) 0.067 mg/ml phenol red in 50 mM sodium phosphate buffer (pH 7.4), and (b) 1 mg/ml horseradish peroxidase in 50 mM sodium phosphate buffer (pH 7.4).

A reaction mixture was then prepared by mixing 1.5 ml of the phenol red solution, 50 μl of 0.1 M H₂0₂ and, after a spectrophotometer trace was taken, 15 μl of the horseradish peroxidase solution. Another spectrophotometer trace of the 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 A is the pre-oxidation trace taken before the peroxidase 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 green, but differs in that before oxidation, the "yellow" (actually orange) peak near 430 nm is quantitatively much larger than the oxidatively reduced "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 at about 405 nm. Similarly, bromcresol purple has a major light absorbance band at about 589 nm and another at about 400 nm. The absorbances at 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 HO in the presence of peroxidase, the absorbances at

SUBSTITUTESHEET 625 nm and 589 nm are extinguished and, as a consequence, the changes in hue and in absorbance are large. Bromthymol blue functions similarly with absorption maxima at 618 nm (extinguished by oxidation) and 405 nm (not extinguished) . 5

EXAMPLE 2 Solid Phase Visual Assay For Hydrogen Peroxide

Filter paper discs (Whatman No. 1), 6.5 mm in 10 diameter, were impregnated with a 5 μl 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 μl portion of an aqueous solution which contained 0.08 percent of horseradish peroxidase in 60 mM sodium phosphate 15 buffer (pH 7.4), and dried. Finally, each of the discs was impregnated with a 5 μl portion of an aqueous 0.1 N sodium nonanoate solution and dried. The dried discs were placed in wells of standard 96-well matrix microtiter plates, and a 5 μl portion of water or of up to 20 mM hydrogen peroxide 20 in water was titrated onto each disc. After the color 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 25.discs from deep royal blue (0 mM peroxide) through shades of blue-green, 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 μl portions of a solution containing 7.5 mg/ml bromcresol green in 50 mM sodium phosphate buffer (pH 7.4) was simply pipetted into microtiter plate wells, followed by 5 μl of 1 mg/ml horseradish peroxidase and 5 μl of water or of aqueous hydrogen peroxide, except that the orange-yellow hue at high peroxide concentrations was not readily- observable in such 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 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 with liquid phase analytes and reagents as described herein. It will also be 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.

EXAMPLE 3

Liquid Phase Kinetic Assay For Peroxidase

A liquid phase kinetic assay for peroxidase was performed by 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 mixing 1.5 ml of the bromcresol purple solution, 50 μl of the H₂0^' ₂ solution, and 10 μl of solution (b) containing from 0 to 1 mg/ml of the horseradish peroxidase, mixing rapidly, and then monitoring light absorbance at 589 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 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:

It will be noted that, within the conditions and concentrations of peroxidase tested (0-100 μg 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 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 (extinguishment of one of he absorbance peaks) proportional to the peroxidase or the like activity present, provided the concentration of dye is sufficient to saturate the enzyme reaction. While the commonly available commercial "peroxidase" enzyme is that obtained from horseradish roots, numerous other natural sources in plant and animal tissues and microbes are known, e.g., potatoes, white blood cells, and various bacteria, and the enzyme is likely widespread in many other biological materials. Also, many other protein and non- protein substances, e.g., heme, exhibit peroxidase activity though with generally lower speed and substrate specificity than classical peroxidase enzymes.

Qualitatively similar results were obtained when the foregoing procedure was repeated, except that phenol red was substituted for bromcresol purple and the change in light absorbance was monitored at 560 nm instead of 589 nm. The data for phenol red are set forth in Table 2 below:

SUBSTITUTESHEET EXAMPLE 4 Effect of pH

The effects of pH upon the kinetics of peroxidative bleaching 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 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 mg/ml bromcresol green in 50 mM sodium phosphate buffer (pH 6.5),

(c') 10 mg/ml horseradish peroxidase in 50 mM sodium phosphate buffer (pH 6.5); and

(d) 0.067 mg/1 bromcresol green in 50 mM sodium phosphate buffer (pH 7.4),

(d') 10 mg/ml horseradish peroxidase in 50 mM sodium phosphate buffer (pH 7.4).

Four reactions were conducted by preparing mixtures at ambient temperature (ca. 22°) in 2 ml spectrophotometric cuvettes by mixing 1.5 ml of each of the bromcresol green solutions [(a), (b) , (c) and (d)], 10 μl 0.1 M H₂0₂ and 5 μl of each of the horseradish peroxidase solutions, [(a'), (b'.), (C) and (d')]. The mixtures were mixed rapidly, and the light absorbance at 612 nm as a function of time was

ET monitored. Figures 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 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 fastest at pH 5.6 (6-7 OD/min). At pH 6.0, the rate was 2.1

OD/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. 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 applications in various settings and for different purposes.

It will be apparent that many modifications and variations of the invention as herein set forth are possible without departing from the spirit and scope thereof, and that, accordingly, such limitations are imposed only as indicated in the appended claims.

Claims

We claim:

1. A method for determining the presence or amount of peroxidase, hydrogen peroxide, or substances capable of generating hydrogen peroxide or catalyzing the generation of hydrogen peroxide, in a test sample, said method comprising the steps of:

(a) forming a reaction system comprising (i) a sulfonephthalein dye or a phthalein dye having an oxidizably extinguishable absorbance peak in the visible region, and (ii) a test sample suspected of containing an unknown amount of peroxidase, hydrogen peroxide, or substances capable of generating or catalyzing the generation of hydrogen peroxide; and

(b) monitoring the extinguishment of said absorbance peak as a function of said unknown amount present in said test sample.

2. The method of claim 1 wherein said reaction system comprises (i) a test sample comprising an unknown amount of peroxidase, (ii) said dye, and (iii) hydrogen peroxide in an amount sufficient to cause extinguishment of the absorbance peak of said dye, whereby said unknown amount of peroxidase present in said test sample is determined by monitoring such extinguishment.

3. The method of claim 1 wherein said reaction system comprises (i) a test sample comprising an unknown amount of a compound which is capable of undergoing a reaction with oxygen to form hydrogen peroxide, (ii) said dye, and (ii) a peroxidase, wherein the hydrogen peroxide which is generated causes extinguishment of the absorbance

TITUTE SH ET peak of said dye whereby said unknown amount of the compound present in said test sample is determined by monitoring said extinguishment.

4. The method of claim 3 wherein said reaction system comprises a catalyst in an amount sufficient to cause the compound to undergo a reaction to form hydrogen peroxid .

5. The method of claim 1 wherein said reaction system comprises (i) a test sample comprising an unknown amount of a peroxide, (ii) said dye, and (iii) peroxidase, wherein the peroxide causes extinguishment of the absorbance peak of said dye whereby said unknown amount of peroxide in said test sample is determined by monitoring said extinguishment.

6. The method of claim 1 wherein said reaction system comprises (i) a test sample comprising an unknown amount of a catalyst which reacts with a compound in the presence of oxygen to form hydrogen peroxide, (ii) said dye, and (iii) a peroxidase, wherein the amount of hydrogen peroxide which is generated causes extinguishment of the absorbance peak of said dye whereby said unknown amount of the catalyst in said test sample is determined by monitoring said extinguishment.

7. The method of claim 1 wherein said reaction system comprises a sulfonephthalein dye.

8. The method of claim 1 wherein said reaction system comprises a phthalein dye.

9. The method of claim 7 wherein said sulfonephthalein dye is selected from the group consisting of bromcresol green, bromcresol purple, bromcresol blue, bromthymol blue, thymol blue and phenol red.

10. The method of claim 7 wherein said sulfonephthalein dye is bromocresol green.

11. The method of claim 8 wherein said phthalein dye is phenolphthalein.

12. The method of claim 1 wherein said oxidizably extinguishable absorbance peak is from between about 500 nm to about 700 nm.

13. The method of claim 1 wherein said unknown amount is determined by comparing the extinguishment of said absorbance peak with the extinguishment of an absorbance peak of a known standard sample.

14. The method of claim 1 wherein said unknown amount is determined by monitoring the extinguishment of said absorption peak with an instrument.

15. The method of claim 1 wherein said unknown amount is determined by monitoring the extinguishment of said absorption peak visually.

16. A reaction system for determining the presence or amount of peroxidase, hydrogen peroxide, or substances capable of generating or catalyzing the generation of hydrogen peroxide, in a test sample, said reaction system comprising (i) a sulfonephthalein dye or a phthalein dye having an oxidizably extinguishable absorbance peak in the visible region and (ii) a test sample suspected of containing an unknown amount of peroxidase, hydrogen peroxide, or substances capable of generating or catalyzing the generation of hydrogen peroxide, whereby said

SUBSTITUTESHξET absorbance peak is extinguished as a function of said unknown amount present in said test sample.

17. The reaction system of claim 16 comprising (i) a test sample comprising an unknown amount of a compound which is capable of undergoing a reaction with oxygen to form hydrogen peroxide, (ii) said dye, and (ii)' a peroxidase.

18. The reaction system of claim 17 comprising a catalyst in an amount sufficient to cause the compound to undergo a reaction to form hydrogen peroxide.

19. The reaction system of claim 16 comprising (i) a test sample comprising an unknown amount of a peroxide, (ii) said dye, and (iii) peroxidase.

20. The reaction system of claim 16 comprising (i) a test sample comprising an unknown amount of a catalyst which reacts with a compound in the presence of oxygen to form hydrogen peroxide, (ii) said dye, and (iii) a peroxidase.

21. The reaction system method of claim 16 comprising a sulfonephthalein dye.

22. The reaction system of claim 16 comprising a phthalein dye.

23. The reaction system of claim 21 wherein said sulfonephthalein dye is selected from the group consisting of bromcresol green, bromcresol purple, bromcresol blue, bromthymol blue, thymol blue and phenol red.

24. The reaction system of claim 22 wherein said phthalein dye is phenolphthalein.

25. The reaction system of claim 23 wherein said sulfonephthalein dye is bromocresol green.

26. The reaction system of claim 16 wherein said oxidizably extinguishable absorbance peak is from between about 500 nm to about 700 nm.

SUBSTITUTESHEET