CA1086199A - Kinetic assay for alpha-amylase - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

An acid phosphatase assay comprising:
a) performing simultaneous reactions at a pH of from about 4.5 to about 6 which comprise;
(i) reacting an organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate.
p-nitrophenyl phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthnlein monophosphate, and phenol-phthalein monophosphate in the presence of acid phosphatase to release inorganic phosphate;
(ii) reacting maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and .beta.-D-glucose-1-phosphate;
(iii) reacting .beta.-D-glucose-1-phosphate in the presence of .beta.-D-phosphoglucomutase to form glucose-6-phosphate; and (iv) reacting glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogonase and a co-enzyme selected from a group consisting of .beta.-nicotinamide-adenine dinucleotide, .beta.-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof to form the reduced form of said co-enzyme and 6-phosphogluconate; and (b) measuring the rate of production of said reduced co-enzyme, wherein the acid phosphatase being measured is rate-limiting and wherein a non-phosphate buffer is used to control said pH.

Description

~18~;~L99 This application i9 a divl~lon o~ co-pendlng Canadlan Application No. 271,565 filed February l.l, 1977.

Background of the Invention .
This invention relates to reagents and methods or deter~
mining alpha-amylase concentration in aqueous solutions such as ser-um and urine.
Alpha-am~lase is an enzyme produced by the human body and is found in fluids such as blood, urine and saliva. It is not entire-ly certain what part of the body produces alpha-amylase, bùt ik is clear that when the body is healthy, the concentration of alpha-amylase present in human fluids will vary over a range of valu~s, and when the body is sufering Erom certain path~logical conditions the alpha-amylase concentration will be higher or lower than the range existing when the body is healthy. For example, when a per-son has pancreatitis, mumps, or pancreatic cancer, the alpha-amy-lase concentration will be much greater than its level in the ab-sence of such condi~ions. Liver diseases may pro~uce alpha~amylase concentrations that are lower than otherwise.
Techniques or determining alpha-amylase conc~ntrations gen-erally involve the use o starch because o~ the catalytic effect of alpha-amylase on the hydrol~sis of the 1,~ linkages of the amylose and am~lopec~in ~ractions of starcll. I this hydrolysis is let to go to completion, the alpha-amylase will pro~ressiveLy deyrade the starch into gluc~se, maltose, and oligosaccharides. Certain tech-niques have attenpted to correlate the decrease in the turbidity or visCosity o an aqueous starch solution ater amylose hydrolysis with the resultant alpha-amylase concentration.
Other techniques utilize the quantity of reducing su~stan-.
ces produced by the alpha-amylase-starch reaction as a measure of j ,j ~ , :
;

" ~8619~
alpha-amylase concentration, or utilize the rate of dye r~lease from a dyed starch by alpha-amylase as a measure of alpha-amylase concentration.
Enzymatic ~echniques have al~o been developed to measure alpha-2mylase concentration by Using alpha-amylase and other en~
zymes to hydrolyze starch into glucose which is then measured through coupled enzymatic reactions. This approach, however, is not ~ ;
satisfactory ~ecause of the presence in many assay specimens of glucose which will react through the coupled enzymatic reactions to produce easily detectable proauct in addition to that produced by enzymatic starch hydrolysis. The concentration of this endogenous glucose is generally significant with respect to the amount of g~u-cose usually produced by the enzymatic hydrolysis technique; and as a result, such pre-exist.ing glucose must be eliminated from the assay specimen before the assay is conducted.
Another technique is the iodometric method which utilixes the well known reaction between iodine and starch to form a blue color. When a blue colored starch-iodine solution is hydrol-~zed with alpha-amylase, the blue color decreases as the alpha-amylase degrades the starch. The change in color of the blue starch-io-dine solution is thus ~ome measure of alpha-am~lase concentration~
This technique, howe~er, has not been considered reliable or su~-iciently defi~ite because it is believed that the change in color ~oes not bear a linear relationship to the concentration of alpha-amylase.
All of the foregoing techniques, while sufficient to yi.elda general indication of alpha-amylase concentration, are not en-tirely satisfactory because they either do not lend themselves to precise scientific measurements and/or are too time-consuming.
Summary of the Invention It is therefore an object of the present invention to pro-vide a ne~ set of reagents and a new method for using these .ea-.

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gents which will overcome the problems associated with prior tech-niques or de~ermining alpha-amylase concentratiOns.
Another ob ject of the present invention is to provide a ne~
procedure for determining alpha--amylase collcentrations which can be S performed quickly, simply, reliably and with reproducible results.
These objects are achieved by the invention disclosed and claimed hereina~er which is a novel kinetic technique for measur-ing alpha-amylase concentrations in aqueous solutions which is based on the following reactions:

(I) alpha-1,4 linked glucan P Y
alpha-maltose ~ other maltooligosaccharides :

alpha-maltose + po4 _ MP ~ glucose ~ beta-D-G-l-P

(III) be~a_D_G_l_p beta-PGM > G 6 (IV) G-6-P ~ NAD G6PDH ~ 6-P-G ~ NADH

wherein the concentration of alpha-amylase in the a~ueous solution is determined by measuring the rate of production of NADH which p~o-~ides a measure of alpha-amylase concentration. The following ab-breviations are employed in the above reactions and hereinafter~
; Abbreviations P04-~ - phosphate ion MP ~ maltose phosphor~lase b~ta-D~GlP - b~ta-D-glucose~l~phosphate beta~PGM - beta-D-phosphoglucomutase G-1,6-diP - D-glucose-1,6-diphosphate G-6-P - glucose-6-phosphate 6-PG - 6-phosphogluconate G6PDH - glucose-6-phosphate dehydrogenase .:
6PDH - 6-phosphogluconate dehydrogenase . NAD - beta-nicotinamide-adenine dinucleotide ~-NADH . - reduced form of beta-nicotinamide-adenine . . dinucleotide The reagent system o~ the present invention con,ains ~he 36~99 startlng material of reactlon I, alpha-1,4 llnked glucan, and all of the constituents except alpha-amylase, needed to make all of the four reactions proceed as indicated, i.e. phosphate ions, MP, beta-PG~, and G6YDH. This reagent system may be provided and used as one mlxture, or it may be provided in a kit consisting of a plural:lty o reagents each of which contains one or more of the :Lngredlents oE the reagent system whlch are al.l mi~ed together when the reagents are used in the alpha-amylase assay of the invention. All of the aforesaid ingredients of said reagent system appear to be stable as one mixture and thus lt is preferred that the reagent system be provided as one mixture inasm~lch as it is easier to work with one reagent .
rather than a plurality.
In one particular aspect the present application, which is a division of aforementioned Canadian Application No. 271,565, is concerned with the provision of a reagent.system for an acid phosphatase assay comprising:
(a) maltos~; , (b) an organic phosphate selected from a group consisting of ~
glycerophosphate, phenol phosphate, p-nitrophenol phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymol-phthalein mono-phosphate, and phenolphthaleln monophosphate;
. ~c) maltose phosphorylase;
(d) a co-enzyme selected from a group consisting of beta-nicotinamide-adenine dinucleotide, beta-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof;
(e) glucoae-6-phosphate dehydrogenase;
(~) beta-D-phosphoglucomutase;
whereln the above are present ln ~he amount such tha~ the nci~
phosphatase to be assayed is rate-limiting, and (g) a non-phosphate containing buffer having a pH from about 4.5 to about 6.
In another particular aspect the present application, which ls a division of aforementioned Canadian Application No. 271,565, is concerned with the provision oE an acid phosphatase assay comprising: ~ :
~ .

jl/b~ ~5~

~ (a) performing si~ultaneous reactlons at a pH of from about 4.5 to about 6 which comprise:
~i) reacting an organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate, p-nitrophenyl phoc~phate, alpha-napht~ly]. phosphate, adenosine-3'-monophosphate, thymolphthaleill monophosphate, and phenol-phthalein monophosphate in the presence of acid phosphatase to release inorganic phosphate;
~ii) reacting maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and ~-D-glucose-l-phosphate;
(iii) reacting ~-D-glucose-l-phosphate in the presence of ~-D-phosphoglucomutase to form glucose-6-phosphate; and (iv) reacting glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogenase and a co-enzyme selected from a group conslsting o~ ~-nicotlnamlde-adenine dlnucleotlde, ~-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof to form the reduced form of said co-en~yme and 6-phosphogluconate; and (b) measuring the rate of production of said reduced co-enzyme, wherein the acid phosphatase belng measured is . rate-limiting and wherein a non-phosphate buffer is used to control said pH. :
~b~ un~ v~n~
Wlth respect to the E:lrst react:Lon employed by the present invention~
(I) alpha-1,4 linked glucan alpha-amylase~ 1 h and other maltooligosaccharides The alpha-1,4 llnked glucan may be any polysaccharide made ~`
up primarily of glucose wherein the glucose molecules are mainly ;
connected through alpha-1,4 llnkages which can be attached by the alpha-amylase. Exemplary of such polysaccharides are starch, amylopectin, amylose, glycogen, dextrin and their degraded jl/ ~\ -5a-.

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products, and homologs oE ~altooligosaccharides such as maltotriose, maltotetraose and maltopentaose or mix~ures thereo~.
Starch is the preferred form of said glucan because it offers the best combination of solubility, low expense, rècovery and stability. Superlose~ 500 is the brand name of a starch which is used in the preferred embodlment of the invention. This starch has good cold water solubility, gives better response and linearity than other starches, ylelds good reproducibility, and is non-turbid in solution. Superlose~
500 is a modified amylose distributed by Stein-Hall Company 1 ;
of New York City. Superlose~ 500 is a white, 5b-.
, , ~ .

6~5~g yranular material having a moisture conten-t of cibout 10 percent, a pH of 7, and a fi ].m t~nsile strength in excess of 8,000 pounds per square inch. The viscosity o~ 5upexlo~e~500 in Broo~field cps at L50F is 185 or 14~ solids, 55 for 10% solids, and 10 ~ox 5% sol-ids. At 75F the viscosity is 2,000 for 14% solids, 275 for 10~
solids, and 30 for 5% solids. Superlos~ 500 dissolves easily in water at room tempera~ure in contrast to most starches which re-~uire some degree of agitation and/or heating before passing into solution. Superlose~500 is made from the modified amylose fraction of potato starch and contains no significant amount of the amylo-pectin fraction of starch.
GR brand starch is the brand name of another starch which may be used in the preferred embodiment. G~ brand starch is dis-tributed by E. Merck Company of 500 Executive Blvd., Elmsford, New York and manufactured by Mexck European of Darmstadt, Germany.
This starch is dialyzed prior to use and has the following charac-teristics: maximum sulfate ash of 7 weight percent; 10% by weight loss on drying; 1 gram of G~starch has a reducing power equivalent ;~
to 7 milligrams of maltose; pH of between 6.5 and 7.5, and a favor-able sensitivi~y test.
According to the present invention, it is necessary thatthe amount o alpha-amylase be rate-limiting. Thus r the amounts of thc other cons~itu~nts of the reagent system of the present inven-tion should be presen~ in suitable amounts to ensure that the ob-served reaction rate for the complete assay system is characteris-tic of and determined by the rate of the alpha-~mylase catal~zed reaction (xeaction I). For the assa~ of aqueous solutions of human ser~m or urine, it is preferred to use a concentration of between about 1.0 to about 20 grams of an alpha-1,4 linked glucan per liter of reagent. A glucan concentration of about 5 grams per liter of reagent is used in the preferred embodiment.
With regard to the second xeaction employed b~ the present .
' ~ -6-` ~)8~; .

invention:

(II) alpha-maltose ~ phosphate M
Glucose ~ beta-D~Glu-l-P
The alpha-maltose produced by the first reaction is reactea 5 with phosphate ions using maltose phosphorylase as an enzymatic catalyst to produce glucose and beta-D-glucose-l-phosphate.
The phosphate ions are supplied from any source compatible with the reagent system o the present invention. I~oxganic phos-phates are an-example of such source. The phosphate used in the preferred embodiment is a mixture of K2~P04 and KH2P04 which ~orms a buffered solution a~ a pH of zbout 6.5 which is optimum.
The concentration of phosphate ions should be at a level to ensure that alpha~amylase i5 the rate-limiting compound. However, it is desirable to have not too high a concentration of phosphate ions because large; concentrations may inhibit the activit~ o the beta-PGM enzyme. It is preferred to have about 0.01 to about 0.1 molar aoncentratian of inorganic phosphate, with about 0.025 molar being the mos~ preferred amount for the assay of serum.
Maltose phosphorylase is an enzyme which catalyzes the reac-tion of alpha-maltose and inorganic phosphate. At least about 200 International Units (IU) of this enzyme per liter o~ reagent is re-~uired, but about ~000 IU per liter is preferred.
The pre~erred source of maltose phosphoxylase is a strain of the microorganism hactobacillus brevis (~TCC8287) which has been cultured by ~eckman Instruments, Inc., Microbics Operations of Carlsbad, California and the enz~me has been extracted and puri-~ied by conventional methods therefrom~ Other sources of this en~
zyme are strains of Neisseria meningitides, Neiseria perflava an~
other Lactobacilli strains.

~egarding the third reaction employed by the present in-vention~

' ' ' . ' ' . . :
.

_7 ~L~)8~195~

~III) beta-D-Glu-l-p beta-PGM > Gl 6 P
The enzyme beta-phosphoglucomu~ase (beta-PGM) catalyzes the conversion of beta-D-glucose-l-phosphate into Glucose-6-phosphate.
Beta-phosphoglucomutase is present in at least about lOO IU per liter of reagent so th~t alpha-amylase o~ reaction I remains the rate-limiting constituent. It is preerred that about 500 IU of beta-PGM per liter of reagent be used when assaying alpha-amylase in human serum. The pre~erred source of beta-PGM is Lactobacillus brevis (ATCC8~87). It is cultured and purified by co~ventional methods of enzyme purification. Other sources include strains of Neisseria ~eningitides, Neisseria perflava and Euglena gracilis.
It is preferred that glucose-1,6-diphosphate (Glu-lt6-diP) be present in the enzyme system to act as a cofactor for beta-PGM. ~ ;~
Beta-PGM re~uires the ~eta-form of Glu-1,6-diP for activit~, but it is believed that the alpha-form o~ this coactor may also wor]c.
The preferred concentration of Glu-l,6-diP should be at least abou~
O.Ol grams per liter o~ reagent. The optimum concentration is about 0.075 gm per liter.
It is also preferred that divalent cations selected ~rom the class consisting of Mn+2, Mg+2, Co~2, zn~2 or Ni~2 be present in the en~yme system to act as a cofactor for beta-PGM. The cat-ions Mn~2, Mg+2, or Co~2 are pre~erred over zn~2 or Ni+~ The cation concentration should be at least about one millimole per li~er o~ reagent and is pre~erably 8~4 millimole6 pex liter.
With respec~ to the fourth reaction employed in the present învention:
~IV) G-6-P ~- NAD G6PDH > 6-P-G ~ NADH
The glucose-6-phosphate is reacted with beta-nicotinamide-aden1ne dinucleotide and G6PDH to produce 6-phosphogluconate and NADH~
The amount o~ NAD should be high enough to ~eep alp~a-amy-.
. . .
8 . : ~ , ~o~ 99 lase the rate-limitin~ consti~uent. A suitable range for the NAD
concentration is about one to ahout 10 millimoles per liter o reagent. The preferred concentration of NAD is about 2 . 5 milli-moles. seta-nicotinamide-~denine dinucleotide phosphate (NADP~ may S be substituted for NAD in the presen~ invention.
The glucose-6-phosphate dehydrogenase ~G-6 PDH) should also be present in a concentration o~ at least about sO0 IU per liter of reagent so that this reaction is not the rate-limiting reaction.
The preerred concentration of the G-6-PDH enzyme is about 5000 IU
per liter of reagent. The preferred source of G-6-PDH is Leucono~
stoc mesenteroides tATCC 122sl) but it may be obtained ~rom other sources. ,-In the preferred embodiment o the present i~ention, it is desirable to employ a fifth reaction as part of the assay:
(V) 6-P-G ~ NAD 6 PD~ ~ ribulose-5-P ~ NADH ~ C02 The purpose o~ this fifth reaction is to increase the sen-sitivity and accuracy of the assay by increasing the amount of NADH
produced.
The minimum concentration of 6-PD~ should be at least about 200 International Units per liter of reagent. The optimum concen-tration of 6-PDH is about 700 International Units per liter. The preferred source of this enzyme is Leuconostoc mesenteroides (ATCC
12291), from whi.ch t~le enzyme has b~en cultured and pur~ied by conventionally known ~et~od~, but it may be obtained from other svurce,s.
Sodium chloride may b~ added td the reagent system to i~-crease the activity of the alpha-am~lase.
Bufers including potassium phosphate dibasic (K2~P04) and monobasic (KH2P04) can be used to obtain the optimum pH in wllich to carry out the reaction sequence. Non-phosphate buffers may he used, but are not preferred because phosphate buffers provide a source of phosphate ions. Exemplary of other buffers which .

il6~99 were tes~ed and found to be satis~ac~ory are piperazine-N, N'-bis (2~ethane5Olfonic acid); tris (hydroxymethyl) aminomethane; N-2-hydroxye~hylpiperazine-Nl-2-ethane su].fonic acid; and triethanol-amine. Exemplary of other buffers whi.ch may also be satisfactory are n-(2-acetamido)iminodi~cetic acid; n-(2-ac~amido)-2-aminoeth-anesulfonic acid; and N,N'-bis~2-hydroxye~hyl)-2 aminoethanesul-fonic acid.
The rate of NADH production and the sonversion of such rate into the concen~ration of alpha-amylase is accomplished by known methods. One such method uses spectrophotometric means to measure : ~he change in absorbance of light due to the production of ~ADH at wavelengths ranging from about 300 to about 370 millimicrons (nm) at a temperature range of from abou~ 15C to about 50C. ~ wave-length of about 340 nm at about 37C is preerred.
When the xate of change in absorbance is measured, the con-centration of alpha-amylase may be calculated ~y the following e~ua-tion wherein the change in absorbance is measured at a wavelength of 340 nm and a temperature of 37~Ci ~ ~A ~ Vt x 1000 IU/liter - V x 6.22 AA = change of absorbance/minute Vt = total reaction volume V8 ~ volume o s~mple containing alpha-am~lase 6.22 = millimolar absorptivity index of NAVH at 340 nm EX~MPLE 1 - INGREDIENTS OF ASS~Y MIXTURE FOR ALPHA-AMYLASE
The following is the composition of the preferred reagent of the pr~sent in~ention prepared as a 1 liter solution of deion-ized water: ~:
Superlose~500 5.00 grams Potassium Phosphate 2.65 grams . - ;
~ibasic i 30 Potassium Phosphate 1.33 grams MonQbasic Maltose Phosphorylase 2000 IU
" , "`, ' ' , , ' .
--10-- , . .

9~
Be~a-Phosphosglucomutase 500 IU
NAD-4H20 1.8 grams Glucose-6-Phosphate 5000 IU
Dehydrogenase 6-Phosphogluconate 700 IU
Dehydrogenase MgC12 6H2 1.7 grams Sodium Chloride 0.5 grams G-1,6-diP 0.075 grams The pH is adjusted to about 6.0 to about 7.5 with a pH of 6.5 being pre~erred .
The reagent system of the present invention may be stored and used in the form of an aqueous solution or the solution may be freeze dried by conventional means and reconstituted with water when ready for use. The reagent s~stem may also be prepared using the constituents thereo in powdered form which are solubilized with water when ready for use.
The enz~me beta-amylase, found in plants as opposed to alpha-amylaee which is found in animals, catalyzes a reaction similar to the first reaction (I), hydrolyzing alpha-1,4 linked glucan to beta-maltose.
In the case of a reagent system for a beta-amylase assay the only modification to the reagent system disclosed hereinabove woul.d be the addition of the enzyme mutarotase to catalyze conver-sion of beta-maltose into alpha-maltose. The kinetic beta~amylase assay therefore comprises the following simultaneous reactlons:

~VI~ alpha-1,4 linked glucan b ~ > beta-maltose ~II) beta-maltose mutarotase > a1pha_maltOse (II) alpha-maltose ~ po4 MP > glucose + beta-D-G-l-P

(III) beta_D_G_l_p beta-pGM > G 6 (IV) G--6-P + NAD 6PD > 6-P-G + NADH

and, in a preferred emhodiment, the following reaction also:
. .

i,199 (V) 6-P-G ~ NAD 6 PDH ~ ribulose-5-P ~ NADII ~ C02 In the above kinetic assay ~o~ beta-amylase, it is necessary that the amount of beta-amylas~ b~ rate-limiting. The amount o~ the var--ious reagents present in the beta-amylase reagent system is the S same as that discussed in the case o~ the alpha-amylase reagent sys-tem with the only difference being the addition to said beta-amyl-ase reagent system of at least about 2000 units, preferably about ~
- 60,000 units, of mutarotase per liter of reagent. ~ ~-~he principles of the instant invention may also be applied to assays for phosphatases and for inorganic phosphate by omitting the constituents of the reagent system of the present invention which ~ake the first reaction proceed and by making the following - additional modifications to the réagent system:
In the case of both the kinetic reagent system and the end-point reagent system or an inorganic phosphate assay, starch and phosphate ion are omitted from the reagent systems and maltose is added to the reagent systems. Both the kinetic inorganic phosphate ~`
assay and the end-point inorganic phosphate assay are based on the following reactions:

(II') m,lltose + po4 - MP > glucose + beta-D-G-l-P

(III) beta-~_G_l_p b-eta-pGM > G 6 P

~IV) G-6-P ~ NAD G6~D~ > 6-P-G ~ NADH

In a preferred embodiment of the kinetic inorgania phosphate assay, the following reaction-can also be employed:

~V) 6-P-G ~ NAD 6 PDH > ribulose-5-P ~ NADH ~ CO2 In the case of the end-point inorganic phosphate assay, it is nec- `;
essary that ~altose and NAD (and NADP, if used) be present in mo-lar excess of the inorganic phosphate to be assayed. In the case o~ the kinetic inorganic phosphate assay, it is necessary that the amount of inorganic phosphate be rate-limiting.
In both the kinetic and end-point assays, the amount of 8~i199 phospha te ion is d~termined b~ measuring thf~ production o NADH, NADPH, or mi.xtures thereof produced by the coupled enzyme reactions ~f the instant inveIItioll. In particular, in the case of the end-point inorg2nic phosph~t~ assa~, the ~noun~ o phosphate ions is determined by measuring the tot~l quantity of N~DH, NADP~I r or mix-tures thereof produced by the coupled enzyme reaction o~ the in-stant invention; and, in the case the kinetic inorganic phosphate assay, the amount of phosphate ions is determined by measuring the rate of pro~uction of NADH, NADPH, or mixtures thereof produced by the simultaneous coupled enzyme reactions of the instant inven-tion.
Both ~he kinetic inorganic phosphate assay and-the end-point inorganic phosphate a~say can be run at a pH o from about 6 to about 8. Preferably, the ~inetic inorganic phosphate assay is run at a pH of from about 6.5 to 8 and more pre~erably at a pH of about 7.4. The preferred pH for the end-point inorganic phosphate assay is about 7Ø

.. . .
~ In both the kinetic inorganîc phosphate assay and the end~
point inorganic phosphate assay, the reagent system can be bufer-~ ed by any non-phosphate buffer having a pH of ~rom about 6 to about 8 and which is compatible with the reagents being used. Exemplary o~ such non-phosphate buffers include; piperazin-N,N'-bis~2-eth anesul~onic acid), N,N'bis(2-hydroxyeth~ 2-aminoethan~ ~ulfonic acid, triethanolamine~ L, and tris~hydro~ymethyl)aminomethane.
~5 N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid is the preer-red buffer for use with the kinetic inorganic phosphate reagent system and piperazin-N,N'~bis(2-ethanesulonic acid) is the pre-ferred buffer for use with the end-point inorganic phospha~e rea-gent system. The reagent system for the kinetic and end-point in-organic phosphate assays are set forth in e~amples 2 and 3, re-spectively.
EX~MPLE 2 - INGREDIENTS OF KINETIC ASSAY MIXTURE FOR INORGANIC
PHOSPHATE

.g~

Preferred Minimum Ingredients Amount Amount ~equired Non-phosphate Buffer 50 mM 10 mM

Maltose 13.9 mM 2 mM

Divalent Cation 2 mM O

Co-enzyme (NAD,NADP) 2 mM 0.1 mM

Maltose Phosphorylase 1.6 IU/ml 0.5 lU/ml ~-Phosphoglucomutase 0.4 IU/ml 0.1 IU/ml 6-Phosphogluconate DH 0.7 IU/ml 0.1 IU/ml Glucose-6-Phosphate DH S IU/ml 1 IU/ml Glucose 1,6-diP 0.05 mM O

EX~MPLE 3 - INGREDIENTS OF END-POINT ASSAY MIXTURE FOR INORGANIC

PHOSPHATE

Preferred Minimum Ingredients Amount Amount Requlred Non-Phosphate Buffer 50 mM 10 m~1 15 M~ltose 13.9 mM 2 mM

Co-Enæyme ~N~D, NADP) 1.6 mM 0.1 ml Divalent Cation 2 mM 0 Glucose 1,6-diP 0.05 mM O

Maltose Phosphorylase 3 IU/ml 0.5 IU/ml 20 ~-Phosphoglucomutase 0~6 IU/ml 0.1 IU/ml Glucose-6-Phosphate DH 5 IU/ml 1.0 IU/ml In the case o~ a reagent system for a~ acid phosphatase a~say, starch and phosphate ion are omi~ted rom the reagcnt and an oxganic phosphate and maltose are included in the reagent. ~lthough any organic phosphate can be used, the organic phosphate is pre-exably select~d from a group consisting of beta-glycero-phos~hate, .
phenyl phosphate, p-nitrophenyl phosphate, ~-naphthyl-phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phe-nolphthalein monophosphate, and more preferably is alpha-naphthyl-phosphate. The kinetic acid phosphatase assay therefore comprisesthe following simultaneous xeactions:

': ' . , ':

.. ,. . . ,, .,, " .. . . . . . .

~ 9 (VIII) organic phosphate (R-PO~ ~ > po~ ~ + R

(II') maltose ~ po4 M ~ glucose ~ beta-D-G-1-P

(III) beta-D-G-l-P _t PGM ~ G-~-P

(IV) G-6-P -~ NAD G-6PDH > 6-P-G + N~DH
and, in a preferred embodiment, the following reaction also:

(V) 6-P-G ~ NA~ 6 PDH > ribulose-5-P ~ NADH + CO2 In the above kinetic assay for acid phosphatase, it is necessary that the amount of acid phosphatase be rate-limiting. The organic phosphate is hydrolyzed by acid phosphatase to phosphate ion. The rate of phosphata ion release is'then determined by measuring the rate of NADH, NADPH, or mixtur~s thereof produced utilizing the coupled enz~matic reactions of the instant invention. The pH of ~he acid phosphatase assay is maintained within a range o from about 4 to below 7, preerabl~ rom about 4.5 to about 6, and more preferably from about 5 to a~out 6. The reagent system can ~e buf-fered by an~ non-phosphate buffer having a pH of from about 4 to below 7 and which is compatible with the reagents being used. Ex-emplary of such non-phosphate buffers are sodium citrate, sodium 'hydso~en maleate,'and sodium cacod~late. (Sodium citrate is tho preferred buffer for use with the kinetic acid phosphatase reagen~
system.) The acid phosphatase reagent system is se-t forth in 'Example 4.

EXAMPLE 4 - ING~EDIENTS OF ASSAY MIXTURE FOR ACID P~OSPHATASE

Pre~erred Minimum Amount ~r d ents Range Re~uired 25 Organic Phosphate 1-5 mM 0.5 mM

Maltose 5-20 mM 2 mM

Maltose Phosphorylase 1-5 IU/ml 0.5 IU/ml ~-Phosphoglucomutase 0.3-2 IU/ml ~ 0.1 IU/ml Co-Enzyme (NAD, NADP) 0.2-4 mM 0.1 mM
. . . ' , ; -15-. . : ~ , . . .
.

i19~

Glucose-6-phosphate Dll 2-10 IU/ml 1 IU/ml Divalent Cation 1~5 mM 0 Glucose-1,6-Diphosphate 0.02-0.2 mM O
Non-Phosphate Buffer 0.02-0.05 M 0.01 M
While the particular embodiment o~ the invention chosen ~ :
herein for purposes o~ the disclosure is at present considered to be preferred, i~ is to be understood that the invention is intended to cover all changes and modifications in the disclosed embodiments which ~all wi~hin the spirit and scope of the invention.

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Claims (11)

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

1. A reagent system for an acid phosphatase assay comprising:
(a) maltose;
(b) an organic phosphate selected from a group consisting of .beta.-glycerophosphate, phenol phosphate, p-nitrophenol phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymol-phthalein monophosphate, and phenolphthalein monophosphate;
(c) maltose phosphorylase;
(d) a co-enzyme selected from a group consisting of beta-nicotinamide-adenine dinucleotide, beta-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof;
(e) glucose-6-phosphate dehydrogenase;
(f) beta-D-phosphoglucomutase;
wherein the above are present in the amount such that the acid phosphatase to be assayed is rate-limiting, and (g) a non-phosphate containing buffer having a pH from about 4.5 to about 6.

2. The reagent system of Claim 1 wherein said buffer is selected from a group consisting of sodium citrate, sodium hydrogen maleate and sodium cacodylate.

3. The reagent system of Claim 1 further comprising glucose-1,6-diphosphate.

4. The reagent system of Claim 3 further comprising 6-phosphogluconate dehydrogenase.

5. The reagent system of Claim 4 further comprising a cation selected from a group consisting of Mn+2, Mg+2, Co+2, Zn+2, Wi+2 and mixtures thereof.

6. The reagent system of Claim 5 wherein said glucose-1,6-diphosphate consists essentially of the beta form thereof.

7. An acid phosphatase assay comprising:
(a) performing simultaneous reactions at a pH of from about 4.5 to about 6 which comprise:

(i) reacting an organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate, p-nitrophenyl phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phenol-phthalein monophosphate in the presence of acid phosphatase to release inorganic phosphate;
(ii) reacting maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and .beta.-D-glucose-1-phosphate;
(iii) reacting .beta.-D-glucose-1-phosphate in the presence of .beta.-D-phosphoglucomutase to form glucose-6-phosphate; and (iv) reacting glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogenase and a co-enzyme selected from a group consisting of .beta.-nicotinamide-adenine dinucleotide, .beta.-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof to form the reduced form of said co-enzyme and 6-phosphogluconate; and (b) measuring the rate of production of said reduced co-enzyme, wherein the acid phosphatase being measured is rate-limiting and wherein a non-phosphate buffer is used to control said pH.

8. The acid phosphatase assay of Claim 7 wherein said .beta.-D-glucoge-1-phosphate is reacted in the presence of .beta.-D-phosphoglucomutase and glucose-1,6-diphosphate to form glucose-6-phosphate.

9. The acid phosphatase assay of Claim 8 wherein said .beta.-D-glucose-1-phosphate is reacted in the presence of .beta.-D-phosphoglucomutase, glucose-1,6-diphosphate, and a cation selected from a group consisting of Mn+2, Mg+2, Co+2, Zn+2, Ni+2 and mixtures thereof to form glucose-6-phosphate.

10. The acid phosphatase assay of Claim 9 further comprising:
reacting 6-phosphogluconate in the presence of said co-enzyme and 6-phosphogluconate dehydrogenase to form the reduced form of said co-enzyme and ribulose-5-phosphate.

11. The acid phosphatase assay of Claim 10 wherein said simultaneous reactions are performed at a pH of from about 5.0 to about 6.