CA1072428A - Fluorometric technique for determining isoenzyme concentrations - Google Patents

Abstract

Abstract of the Disclosure A substrate medium subsequent to its removal from a sup-post means containing a separated isoenzyme is contacted with a reagent selected from a group consisting of a composition compris-ing from 90 to 100 weight percent alcohol per unit volume, said alcohol containing from one to six carbon atoms, and from zero to 10 weight percent urea per unit volume; ketones containing from three to eight carbon atoms; an inorganic salt solution comprising from about 90 to about 99.99 percent of a solvent selected from a group consisting of water and inert polar organic solvents and from about 0.01 to about 10 percent of an organic salt having a formula R(Y)2 wherein R is selected from a group consisting of Pb+2, Ca+2, Sr+2 and Ba+2 and wherein Y is selected from a group consisting of Cl-, N03-, and C104-, and mixtures thereof, so that the fluorescent product present in the substrate medium is preci-pitated in situ, thereby localizing its presence and increasing the fluorometric detection technique's sensitivity.
When creatine phosphokinase enzyme is electrophoreti-cally separated into its isoenzyme constituents, the above fluor-ometric detection technique is further improved by employing an electrophoretic buffer comprising an acid having a formula HOOC(CH2)nCH(NH2)COOH wherein n is an integer from 1 to 4, tris-(hydroxymethyl)aminomethane, and water. This buffer has a pH of 7 to 8.5 at 23°C. and an ionic strength of 0.02 to 1.5.

Description

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'l'hi~ )1ic,ltic)~ .l (livision of Canadian Application No. 263,124 fil~l Oct~b~r 12, 1976.

l~ackqroun(l o.~ t:he Inverlt:ior 1. E`~eld oF the rJlv~JI~ion Thi.s illven~ion pertains to a fluoxometric ;nethod for determini~lc~ ~he illdividua] activ:ities o isoenzymes and to an S. electrophoret;.c method ~or separating crea-tine phosphokinas~.

2. Description of the Prior ~rt , . .. .
. Fluorome-tric techniques for cletexmining the presencc o~
isoenzymes ar~ well known to those skilled in the ar~. A. L.
Sherwin et al , "Eluorescent Technique to Demonstrate Creatine 10. Pshosphokinase ~sozymes", Clinica Chemica Acta, 17: 245 to 249 (1967). The standard fluorometric technique employed ~y those skilled in the art entails detectiny the presence of the NADH, NADPH, derivatives thereo~, and mix-t~lres thereof (hereinafter re-ferred to as the "fluorometric product") in an electrophoretic 15. medium~ H. Somer et al., "Demonstration o Serum Creatine Kinase Isoenzyme by Fluorescence Technique", Clinica Chemica Acta, 40:
133 to 138 (1972); D. W. Moss e~ al., "Characterization of Tissue Alkaline Phosphatases and their Partial Purification by Starch-Gel Electrophoresis", Biochem. ~ , 81: 414 to 447 (1961); and F. R.
20. Elevitch, "Fluorometric Techniques in Clinical Chemistry", Little, Brown and CompanyJ Boston, Mass., ~1973). Several problems are inherent in detecting th~ fluorometric product in the electro-phoretic medium. These problems include the presence of an albu-min artifact (J. Goldberg, "Enzymes in Medicine", Vol. 7, No. 3 25. 526 (1973); ~lelena Update, Vol. 18 (Augus-t, 1973); and R. Wong et al., "Cellulose Acetate Electrophoresis of Creatine Phosphokinase Isoenzymes in the Diagnosis of l~'yocardial Infraction", A. ~ C
P , 64: 209 to 216 (1975~; the presence o~ beta-lipoprotei]l arti~
facts (C. X. Xowe et al., "Combined Isoenzyme ~nalysis in the 30. Diagnosis of ~Iyocardial Injury: ~pplicatioll o~ Electrophoretic ~lethods for the Deteclion and ~uantitat.ion of ~he Creatine Pshos-pshoki.nase ~l~ Isoenzyme", J. Llh. _lin. Iv~ed., Vol. 8, No. 4, 577 to 590 (1972); ~he interFcrence -in ~1uorometric detection by drugs (F.R. Elevitch, su~rcl); and inaccuracies cclused by the diffusion of bands (Rowc et al, supra, and Wong et al, su~ra).
~nother problem present in prior art fluorometric methods for detecting fluorometric products is the well known fad:ing problem, i.e., the disappearance of some of tlle fluorescence of the bands during drying. See H. Somer, et al, supra.
The detection of isoenzyme products in the substrate medium is well known in the art of colorimetry. See S.S. Kind, "Stable Test-~apers for Seminal Acid Phosphatase", Nature, Vol.
182, No. ~646 (Nov. 15, 1958); J. Clausen, "Immunochemical Tech-niques for Identification and Estimation of Macromolecules", North-Holland Publishing Co., Amsterdam (]969), and G.J. Bremer and C.F. Singer, "An Introduction to Isoenæyme Technique", Academic Press, New York, N.Y. Fluorometric methods wherein fluorometric products are detected in the substrate medium are also known. See Ger. 2,136,880.
The diffusion and fading problems have both been a significant defect in fluorometric determinations of isoenzymes since the introduction of the fluorometric method for detecting isoenzymes in about 1972.
The present invention has overcome the diffusion problem via the discovery that by precipitating the fluorescent product in filter paper or other suitable substrate medium, the bands are localized and the sensitivity of the fluorometric technique thereby increased. Further, the instant invention also encom-passes the disco~ery that the fading phenomenum can be greatly curtailed by dehydrating the substrate medium via solvent replace-ment.
SUMMARY OF T~[E INVENTION
In one aspect the parent application, Canadian Applic-ation No. 263,124 filed October 12, 1976, is concerned with the jl/~' ~l -3-. . .

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provisioll nr In improveLI e~oc~ro~)hc)r~tic Ic~cllniqlle ror separating cre~tine phospllok:inase into its const:Ltnent :isoen~ymes oE the type wherein a s~ml)le cont.-l-ini.ng creatine phosl)holinnse :is applied to an e]ectrophoretic med:ium, sa:id e]ectroplloretic medium is placed into an elc~troplloretic cell having located therein an electrophoret:ic buffer in contact with two electrodes, and saicl isoenzymes are separated by applying a direct electrical current to the electrophoretic medium, wherein the improvement comprises using as said electrophoretic buffer one comprls-lng (a) an acid having a formula IIOOC(CII2)nCll(NH2)COOH wherein n is an integer from about 1 to about 4, (b) tris(hydrooxymethyl)aminomethane, and (c) water, said electrophoretic buffer having a pll range of about 7 to about 8.5 at 23C and an ionic strength of about 0.02 to about 1.5.
The present application, a division of aforementioned Canadian Application No. 263,124, is particul.arly concerned with the provision of in a method for measuring isoenzyme concentrations wherein enzymes are placed on a supI;or-t means and separated into isoen~yme con-stituents by separating means, said support means containing said separated isoenzymes is contacte-l w:ith a substrate medium including a substrate and a stabili~ing medium, the substrate medium and support means are separated, and a Eluor.escent product is fluorometrically detected in the substrate medium, the improve-ment comprising: contacting said substrate medium subsequent to its separation from the support means and prior t.o the fluorometric detection with a reagent selected from a group cons:isting of a composition comprising from 90 to 100 weight percent alcohol per unit volume, said alcohol conta-ln:ing from 1 to 6 carl~on atoms, and from 0 to 10 weight percent urea per unit volume; ketones containing from 3 to ô carbon atoms; an inorganic salt sol~ltion comprising from about 90 to about 99.99 percent of a solvent selected form a group consisting of water an(l inert pol.ar orgall:;c jl/ -3a-7'~
, ~
Sl)lvell~S alld frul~l U~ lt 0.01 t(~ about 1() percent of an inorganic salL llavi-lg ., ~ormul~ (Y)2, whereill R i~ s~lected ~rom a group consisting Or l'b Z~ Ca 2, Sr 2 and Ba 2 and wherein Y is selected from a group consistin~ of Cl , NO3 , ancl C10~, , and mixtures thereof, so that the fluorescent product is precipitated in situ in t~e substrate medium therehy localizing its presence and increasing the fluorometr:ic techniclue's sensitivity.
BRIEF DESCR_PTION OF l'llE DR~WIN~
Figure 1 is a comparison of the electrophoretic separation of creatine phosphokinase obtained when using a tris-a~spratate buffer vs. a barbital buffer.
DESCRIPTION OF l'HE PREFERRED EMBODIMENTS
The method of the present invention can be used to measure the concentration of any isoenzyme capable of directly reacting to produce a fluorescent product or of be:ing part of a reaction sequence which produces a fluore~scent product. E~emplary enzymes having isoenzymes capable of fulfilling the above require-ment include creatine phosphokinase, lactate dehydrogenase, jl/' -4--2~

ylucose-6-pllosphate deh~dro(Jen~se, hexokinase, mala~e dehydroge-nase, isocitrat~ dehyd~o~nase, aldolase, and a]cohol dehydroge-nase. ~3ecause of their common use in clinical laboratories, crea-tine pllosphokin~xe and lactate dehydrogenase are preferably used in the present invention.
The general anal~sis of isoenzymes can be broken down into three basic s~eps. Step 1 is the placement of the sample containing an enzyme onto a support means and physically separat-ing the enzyme into its various isoenzyme components by any of the 10. various separation means well known to thbse skilled in the art.
Step 2 is the location of each isoenzyme component. This consists of a set of chemical reactions catalyæed by the isoenzyme compon-ents to produce a detec~able chemical product, such as a fluores-cent product, the quantity of which is in proportion to the amount 15. O~ isoenzyme present. Step 3 is a quantitation of each isoenz~ne component. This lS accornplished by scanning either the support means or the substrate means with a fluorometer or densitometer to quantitate the chemical product produced by each isoenzyme component 20. Various means of sepàrating isoenzymes are well known to those skilled in the art. These methods include gel electro-phoresis ~cornmonly referred to simply as "the electrophoretic separation method"), thin-layer gel chromatography, thin~layer electrophoresis, and thin-layer isoelectric focusing. The tech-25. nique most commonly used in clinical laboratories at present, and therefore the technique most preferably used with this invention~s improved method for detecting fluorometric produc~s, is the elec-trophoretic separation method. In general, the electrophoretic separation of isoenzymes is accornplished by applying a sample, such as human serum, to an electrophoretic medium. The electro-phoretic medium is placed into a chamber known as an electro-phoretic cell which contains an aqueous buffer known as an -5~

372~

electrop~loreLic buf~c~r. Tile electrop}loretic medium is p]aced in direct contact ~ith tlle el~ctrophoretic buffer and in turn the electl^ophoretic buffer is in clirect contact wi-th t~Jo sep;~r~te elec~rodes (a cathode and ~n dnode)~ The e~lec~rodes are connected 5. with a direct current power source whereby the electrphoretic process takes place when direct current is applied to the elec-trophoretic medium. In order to adequately separate the various isoenzyme constituents, the electrophoretic process usually takes place at an established electrical potential, for example, from about 50 to about 500 volts, and for a sufficient length of time, for example, from about 0.1 to about 2 hours.
The location of each isoenzyme component requires con-tactiny the separated isoen~ymes with reagents capable of reacting to produce a detectable product. One method weIl known to those 15. skilled in the art encompasses contacting the separated isoenæymes ~ith a substrate medium containing said reagents. The substrate medium is prepared by incorporating the specific chemical sub-strate required for the specific isoenzyme system into a stabiliz-ing medium such as filter paper, chromatography paper, cellulose 20. ace~ate membrane, agarose gel, etc. The preferred stabilizing medium for use in the present invention is filter paper and chro-matography paper, the latter being the stabilizing medium of choice. To the stabilizing medium is usually added a substrate buffer, metal ions as activators, specific reactants or sub-25. strates, and a co-enzyme, i.e.l N~D, NADP, derivatives thereof, and mixtures thereof, which serves as a fluorogenic substrate.
After electrophoresis, the electrophoretic medium, as well known to those skilled in the art, is placed into a chamber and the substrate medium is placed in direct contact witl-l the sur-30. face of the electrophoretic medium. The electrophoretic and sub-strate media are incubated at a predetermined temperature and time. Although tl^~e incubation temperature and time are not ~7~

critica], the incubation proc~ss use~d in the preserlt invention preferably takes place at a temperature of ~bout 20 to about 50C., more pre~crably at a telllperclture of about 35 to about 39C
and preferably ~or a period o~ time from about 30 to about 90 minutes and more preferably for a period of time fr~m abollt 45 to about 75 minutes. During incu~ation, the chemical substrate dif-fuses into the electrophoretic gel where the isoenzyme reacts upon the substrate to produce, i.e., catalyze, the formation of specific fluorometric products. The fluorometric products formed lO. in the zones where isoenzymes are located are free to diffuse back into the substrate medium. Proteins, lipids, and bound drugs do not diffuse any appreciable amount into the substrate medium because of their large physical size, i.e., molecular weight.- ~fter termination of the incubation phase, the substrate 15. medium is separated from the electrophoretic medium.
Detection of the fluorescent product is accomplished by exciting the fluorescent product with ultraviolet light in the range of 300 to 400 nm with maximum absorbance at 340 to 350 nm.
The fluorescent product fluoresces with a visible light at 400 to 20. 500 nm with a maximum fluorescence about 460 nm. Detection of the visible fluorescent light can be done visually with a fluorescent densitometer or a fluorometer. Another detection technique which can also be employed comprises cutting out the areas of the sub-strate medium which contain the fluorescent product and placing 25. each section into an aqueous solution to redissolve and elute the fluorescent product from the substrate medium. Once this has taken place the e]uted fluorescent produc~ can be measured by ab-sorbance, fluorescence, or by a chemical assay method. The a-mount and position of the fluorescent product detected is pro-portional to the isoenzymes located in the electrophoretic mediumznd therefore to the amount of isoenæymes in the sample being assayed.

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- T~o olher e~emplary methods for d~tecting the fluoro-me-tric produc~ are ~l~e pho~ographic reproduction technique and the photo~ aphic negative scannillg technique. The former is a qualitative method which involves visually examining a picture 5. taken of ti~e substrate Inedium. The latter is a quan~itative method whic~l entails densitometrically analyzing ~he negative of a photograph taken of the substrate medium.
The improved fluorornetric detection technique within the scope of the present invention entails modifying the above 10- isoenzyme analysis steps so that prior to the fluorometric detec-tion step the fluorometric product is precipitated in situ in the substrate medium. The precipitation of the fluorescent product can be brought about by the use of a reagent selected from a group consisting of a composition comprising from about 90 to 100, pre-15- ferably 100, weight percent alcohol per unit volumej the alcohol containing from one to six, preferably from one to three, carbon atoms, and from zero to 10, preferably zero, weight percent urea per unit volume; ketones containing from three to eight carbon atoms, preferably from three to five carbon atoms; an inorganic salt solution comprising from about 90 to about 99.9, preferably from about 98 to about 99, percent of a solvent selected from a group consisting of water and inert polar organic solvents, pxef-erably ~rom the group consisting of alcohol containing one to six carbon atoms and ketones containing three to eight carbon atoms~
25- and more preerably from the group consisting of alcohols con-taining one to three carbon atomg, and from about 0.01 to about 10, preferably from ahout 1 to about 2, percent of an inorganic salt having a formula R(Y)2, wherein R is selected from the group consisting of Pb+2, Ca 2, Sr+2, and Ba~2 and wherein Y is selected 30. from a group consisting of C1 , N03 , and C104 ; and mixtures thereof. Other exemplary inert polar organic solvents include z~

dimet}lyli~o~ amide a~l~ dim~t~ly1acetamicle. The sole criteria that the il~rt polar or(3anic so]vents must satisfy is that they must be ab1e ~o solubiliz~ ~he inoryanic salt. The p~eferr~d reay~nt is isop~opanol. The a),ove ~acJents can be used along ~lith a poly-5- mer addition, e.CJ., polyprop~]ene glycol , polyethylene glycol, etc., to enhance precipitation and control surface characteris-tics (i.e., to prevent warping or wrinkling) of ~he substrate medium.
It is also desirable to dehydrate the substrate medium 10- to increase the intensity of the fluorescence of the fluorescent product. The dehydration can be accomplished by evaporation of the water from the substrate medium or preferably by solvent re-placement such as alcohol and ether replacement of water4 To eliminate the previously referred to undesirable lS- fading phenomenum, it is preferred to combine the precipitation and dehydration into a one step process by placing the substrate medium into alcohol for a short period o~ time. At this point the fluorescent product can be detected by fluorescence, but to ~;
aid handling, the alcohol is preferably allowed to evaporate which 20- results in a dried substrate medium containing a localized, rela-tively non~fading, fluorescent product.
Although there are many suitable electrophoretic buf-fers known to those skilled in the art which can be used in the electrophoretic isoenzyme separation step described above, when 25. one is electrophoretically separating creatine phospholcinase into its constituent isoenzymes it is preferred to use a particular electrophoretic buffer comprising (a~ an acid having a formula HOOC(CH2)nCH(NH2)COOH wherein n is an integer from about one to about four, preferably from about one to about two, (b) tris-30- (hydroY~ymethyl)aminomethane, and (c) water, preferably distilled or deioniæed water and mixtures thereof. This electrophoretic 3L~7Z~

buf~er has a p]l of about 7 to about 8.S, ~referably about 7.2 to about 7.7, and m~re pref~r~bly 7.5, at 23C., ~nd an ionic stren~th oE ahou~ 0.02 to about 1.5, preferabl~ about 0.03 to about 0.07, and more preferably about 0.05.
There are several xeasons why the aforesaid electropho-retic buffer is preferred for use in electrophoretically separat-ing creatine phosphokinase isoenzymes. One reason is that the use of barbital buffers, or other buffers similar to barbital buffers, proauce an application artifac-t. The application arti-10. fact results from the creatine phosphokinase enzyme known as CPK3 being electrophoretically located at the trench sample applica~
- tion point, thereby producing an outline of the application area when the electrophoretic medium is scanned densitometrically.
Also, these prior art buffers electrophoretically separate the 15. creatine phospho]cinase isoenzyme known as CPKl into an area where-in serum albumin is present. This type of separation interferes with the enzymatic action of the CPKl isoenzyme. B. A. Nealson et al., J. Clin. Pathol., 28 ~10): ~34 to 836 (197S). Further, the pH of prior art buffers is in the range of about 7.5 to 9, 20- while the optimum pH for the determination of creat~ne phospho-kinase isoenzymes is 6.8. In contrast with the above disadvan-tages, by using the above described buffer, the sample artifact is eliminated because the CPK3 isoenzyme is located entirely to the cathode side of the sample application point~ Alsor the in-25- terference from serum albumin with the CPKl isoenzyme is also eliminated because this creatine phosphokinase isoenzyme is lo-cated entirely to the anode side o the area occupied by the serum albumln. Further, although the p~ titration curve for the above described buffer is similar to the barbital buffer curve, 30. the lo~er pH of the above buffer as compared to the high pH of the barbital buffer enables one to use less substrate buffer in 10'72428 order to lowc-: tlle pll o~ tlle elec~rophoretic medium to the opti-mal p~ rallge for creatine phosphokillase isoenzymes.
The following e~amples ~re provided for the purpose of further illustration only and are not intended to be limitations 5~ of the disclosed invention.
Example 1 Analysis of Creatine Phosphokinase Isoenzymes by Fluorometric .Detection of Reduced NAD.
I. Electrophoretic Technique 10. 1. ~lectrophoretic buffer: To prepare the electrophoretic buffer, dissolve the following reagents in one liter of - distilled water.
a) 6.7 grams tris(hydroxymethyl)aminomethane . ~ :
b) 6.0 grams DL-aspartic acid .
15. 2. Agarose gel: To. prepare an agarose .gel, one gram of agarose is dissolved in 100 ml of eIectrophoretic buf~
fer with heat.. Apply 12 ml of the warm buffered aga-rose solution to a 2-3/4 x 6-1/2.inch sheet of Mylar brand polyester film (Mylar is a trademark of E. I. du 20. Pont de Nemours & Co., Wilmington, Delawarej which has been coated with .a hydrophilic resin subbing. Place into the warm agarose solution four 1/2 x 1/32.inch steel rods in a linear fashion across the width and ap-- proximately three inches from the end of the Mylar 25~ sheet. After the. agarose solution has solidified to a gel, the .four steel rods are removed with a magnet thereby forming four slots in the gel matrix which .can be used to contain the sample specimens for electro- :
phoretic separation. The gel surface .is gently blotted 30. with a sheet of Whatman No. 2 chromatographic paper before the sample specimens are applied to the gel.

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3. Sample_51 eci,rnen:
a) Serum from a freshly clotted blood b) Body fluid, such as pleural fluid c) Tissue extracts 5, 4. Sample_~pplication: Apply 5 ~1 of sample to each slot in t~le gel with a microliter syringe. Place the gel into an electrophoretic cell which contains electro-phoretic buffer.
5. Electrical Parameters: Perform the electrophoretic 10. separation at 150 volts for a duration of 30 minutes, II. Substrate Overlay Technique 1. Substrate: Use any commercially available creatine phosphokinase reagent.
2. Substrate Overlay 15. a) Saturate a 2-3/4 x 4 inch sheet of Whatman No. 542 chromatograph paper with 1.5 ml substrate solution.
b) After electrophoresis has been terminated, remove the agarose gel from the electrophoretic cell and place it into a small pIastic incubation box.
20. With a sheet of Whatman No. 2 chromatography paper, gently blot the surface by laying the'substrate overlay paper onto the gel surface in a rolling motion to avoid entrapment of air bubbles between the gel surface and the substrate overlay paper.
25. c) Place a lid on the plastic incubation box and incu-bate the combination gel substrate overlay paper at 37C. for 60 minutes.
3. Precipitate and Dehydration Precipitation and dehydration o~ the reduced NAD is ac--30, complished by removing the substrate overlay paper from the 'gel surface and immerging the'substrate overlay .

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paper i31tO 1 no ml 0~ 2-propanol for a period of five minutes. Then p]acing the overlay onto a paper towel, it is allowed to dry.
III. Detectj.on o~ Fluorescence 5. 1. Excitation Source Expose the dried substrate overlay paper ~o a source o~
ultraviolet light in the spectral region of 300 to 400 nm.
2. Fluorescent' Detection . . _ . .
10. a) Direct visual observation b~ Scanning fluorescent densitometer c) Eluorometer d) Pho.tographic reproduction . Example 2 ; 15. Analy~sis of Lactate Dehydrogenase Isoenzy~es by ~luorometric Detection, of Reduced NAD.
I. - Electr,ophoretic Technique 1. Electrophoretic buffer: To prepare the electrophoretic ~ -- .
buffer, dissolve the :~ollowing reagents in one liter of 20. distilled water.
a) 2.6g sodium barbital ' b) 1.15g citric acid c) 3.5g tris-(hydroxymethyl)-amino methane 2. Agarose gel - See Example 1 :~
. .
25. 3. Sample Specimen - See Example 1 - ~ ~.

4. Sample Appl'ication - See Example 1

5. Electrical P'arameters - Perform the electrophoretic s~paration at 150 volts for a duration o~ 45 minutes.
II. Substrate Overlay_Technique 30. 1. a~ 30 mg L-lithium lactate b) 15 mg ~-Nicotinamide adenine 'dinucleoti.de (NAD) c) 1.5 ml electrophoretic buffer ' ~.~7~2L~
2. Suhstrat( Ove~y - Se~ Example 1 ~. l'rec.i~tat:iorl and ~c~llydration ~ ~ee Example 1 III. ~etecti.oll of Fluoresce1~ce - See ~.ample 1 __ _ ~ther enz~ es, for e~ample, qlucose--6-phosphate deh~dro-5 ~nase, he~o]cinasc, malate dellydrogenase, isocitrate dehydroge-nase, aldolase, and alcohol dehydrogenase can be fluoromeLrically analyzed in a manner analo~ous to that set.forth in examp]es 1 and 2.
Example 3 10. In order to demonstrate the ability of various buffers to approach the optimum pH for the determination of creatine phosphokinase isoenzyme, 8 ml of the electrophoretic buffers of examples 1 and 2 were separately mixed with 1 ml of creatine phosphbkinase substrate and the pH of ~he mixture was measured.
: 15. The reswlts derived from these experiments are listed in Table I.

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r `' ` '~ a a " ~7~215 T~b]e I c]ear]y indicates that the electrophoretic bllf-fer wi.thin the scope of this invention, as exemplified by the electroplloretic buf~er of Example 1, more closely approximates the desirable pM of the c~eatine phosphokinase substrate than does 5~ the prior art electrophoretic buffer of Example 2.
:; Example 4 The creatine phosphokinase enz.yme present in a sample of - human serum was electrophoretically separated under an electrical current of 1.~ volts per centimeter for 30 minukes. This separa-10. tion was performed in both a tris-aspartic buffer and a barbital buffer. The barbital buffer comprised 5.1 grams per weight sodium diethyl barbituate and 0.92 grams per weight diethyl barbituric acid. The tris-aspartic buffer employed was the electrophoretic buffer of Example 1. The locations of the various isoenzymes are 15. shown graphically in Figure 1 and numerically in Table II.
The encircled negati~e signs in Figure 1 represent the cathode electrode and the encircled positive signs represent the anode electrodes. The metric scale in Figure 1 is employed to measure the distance that the isoenzymes traveled from the sample 20. trench ~sample application point) as well as the width of the various areas deplcted in Figure 1. CPKl, CPK2, and CPK3 are the three isoenzymes presen~ in creatine phosphokinase and albumin is a protein also present in human serum.
Figure 1 clearly indicates that when creatine phospho-25. kinase is electrophoretically separated using the barbital buffer,the CPK3 isoenzyme is present on both sides of the sample trench, thereby producing an appli.cation artifact. However, when crea-tine phosph4kinase is electrophoretically separated using the tris-aspartic buffer, the CPK3 isoenzyme is only located on the cathode side of the sample trench, thereby eliminatillg the sample artifact mentioned above.

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I igur( 1 1I so c~ c ~ depic~s th~t ~h~n creatine phos-~ okinajc is e.l.e(~.ror?lloretica]]y separated usin~3 thc barbital buffer, the ar.~d occupie~ by ~he albumin ov~rlaps the area occu-pieA by t~le CPKl isoenr~yme. 'rhis ovèr]ap, as noted above, inter-5. feres ~ith the enzym~ti~ action of -the CPKl isoe~zyrne. secause there is no overlap present when creatine phosphokinase is elec-trophoretically separated using the tris-aspartic buffer this interference is totally eliminated.
Table II depicts in numerical form the same information 10. conveyed in Figure 1. The millimeters referred to in Table I
correspond to the metric scale shown in Figure 1.
Fisure I and Table II clearly indicate that the electro-phoretic buffer within the scope of ~his invention, as exemplified by the electrophor.etic buffer of Example 1, is able to eliminate 15. both .the trench artifact and the albumin interference present in the prior art barbital buffer electrophoretic separation.
While various methods and reagents have been described in detail herein, it should be appreciated .that changes and modi-fications may be made without departing from the spirit of the .
20. invention. For example, while the. use of tris-asparti.c buffer . has been described above as being capable of improving the elec-trophoretic separation of creatine phosphokinase wherein the creatine phosphokinase isoenzymes are detected via the present invention's fluorometric technique, it should be clear said tris-25. aspartic buffer can 21so be employed in the electrophoretic sep-aration of creatine phosphokinase wherein the creatine phospho-kinase isoenzymes are detected via prior art f.luorescent and non-fluorescent techniques.

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Claims (4)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a method for measuring isoenzyme concentrations wherein enzymes are placed on a support means and separated into isoenzyme constituents by separating means, said support means containing said separated isoenzymes is contacted with a substrate medium including a substrate and a stabilizing medium, the sub-strate medium and support means are separated, and a fluorescent product is fluorometrically detected in the substrate medium, the improvement comprising:
contacting said substrate medium subsequent to its separation from the support means and prior to the fluorometric detection with a reagent selected form a group consisting of a composition comprising from 90 to 100 weight percent alcohol per unit volume, said alcohol containing from 1 to 6 carbon atoms, and from 0 to 10 weight percent urea per unit volume; ketones containing from 3 to 8 carbon atoms; an inorganic salt solution comprising from about 90 to about 99.99 percent of a solvent selected from a group consisting of water and inert polar organic solvents and from about 0.01 to about 10 percent of an inorganic salt having a formula R(Y)2, wherein R is selected from a group consisting of Pb+2, Ca+2, Sr+2 and Ba+2 and wherein Y is selected from a group consisting of Cl- , N03- , and Cl04- and mixtures thereof, so that the fluorescent product is precipitated in situ in the substrate medium thereby localizing its presence and increasing the fluorometric technique's sensitivity.

2. The improved fluorometric technique of Claim 1 wherein the improvement further comprises dehydrating said substrate medium subsequent to its contact with said reagent and prior to the fluorometric detection.

3. The improved fluorometric technique of Claim 2 wherein said enzyme is selected from a group consisting of creatine phosphokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, malate dehydrogenase, isocitrate dehydrogenase, aldolase, and alcohol dehydrogenase; wherein said stabilizing medium is selected from a group consisting of filter paper and chromatography paper; wherein the contacted substrate medium and support means are incubated at a temperature of about 20° to about 50°C for about 30 to about 90 minutes and wherein said reagent is selected from a group consisting of alcohols containing from 1 to 3 carbon atoms.

4. The improved fluorometric technique of Claim 3 wherein said enzyme is selected from the group consisting of creatine phosphokinase and lactate dehydrogenase; wherein said stabilizing medium is chromatography paper; wherein the contacted substrate medium and support means are incubated at a temperature of about 35° to about 39°C for about 45 to 75 minutes; and wherein the reagent is isopropynol.