DE2731421A1 - PROCEDURE FOR DETERMINING THE AMYLASE CONTENT OF SAMPLES - Google Patents

Description

273H21

FROM KREISLER SCHONWALD MEYER EISHOLD FUES FROM KREISLER KELLER SELTING

PATENT LAWYERS Dr.-Ing. by Kreisler f 1973

Dr.-Ing. K. Schönwald, Cologne Dr.-Ing. Th. Meyer, Cologne Dr.-Ing. K. W. Eishold, Bad Soden Dr. J. F. Fut-i, Cologne Dipl.-Chem. Alek von Kreisler, Cologne Dipl.-Chem. Carola Keller, Cologne Dipl.-Ing. G. Selling, Cologne

5 COLOGNE 1

DtlCMM / .NNMAUS AM HAUPTÜAHNHÜf

July 11, 1977 AvK / Ax

EGG. Du Pont de Nemours and Company, Wilmington, Del., USA

Method for determining the amylase content of samples

709886/0633

Telephone: (0221) 23 45 41-4 - Tele «. g:> 32307 dopo d - kk <jia.nm Dompoicnt Cologne

The invention relates to amylase determinations and a reagent test pack for use therewith Provisions. The invention relates in particular to amylase determinations in which a polysaccharide is used as Amylase substrate is used.

ct-amylase is an enzyme that the α / Τ> 47 ties

hydrolyzed between the glucose units in starch and the lower polymers and oligomers of glucose. This enzyme is produced in the human body, primarily in the pancreas and salivary glands, and its concentration in various body fluids is a valuable diagnostic tool for the doctor. For example, serum α-amylase concentrations are proportionate in healthy individuals constant but increase in response to pathological conditions such as acute pancreatitis.

U.S. Patent 3,879,263 and U.S. Patent Application 542,236 (1/20/1975) describe a method and a combination of reagents for use in determining the α-amylase content of a sample using the defined oligosaccharides maltotetraose, maltopentaose or maltohexaose as an amylase substrate. Preferably by the reaction between a-amalysis and these substrates in the presence of a maltase a certain amount of glucose is formed, which with the help of any usual glucose detection system can be measured. The additional step of detecting the glucose is a disadvantage. Furthermore, glucose present in the sample must either be removed or compensated for.

Although this can be done using conventional methods, this represents an additional step in the process and a disadvantage.

709886/0633

APJansen and PGAB Wydeveld state in Nature 162 (1958) 525 that a- (p-nitrophenyl) maltoside could be suitable as a substrate for the amylase determination. However, this publication shows that the authors never identified the active agent responsible for their observations. They report: 1) By incubation of human urine or saliva samples with <x (p-nitrophenyl) maltoside at 37 ⁰ C for 16 hours 4-nitrophenol was formed, which was spectrophotometrically identified by mixing the hydrolyzate with O, O2n-sodium hydroxide . 2) The hydrolysis was prevented by precipitants for protein, eg 10% trichloroacetic acid and 0.5N silver nitrate. 3) The hydrolysis was pH dependent and was most effective at pH 5.9 to 7.0. The authors state that this was evidence of "the possible presence of an unidentified carbohydrase". It is not believed that a- (4-nitrophenyl) maltoside is for a

in people
Amylase determination / is suitable because the cleavage of this compound by ot-amylase is extremely slow.

The invention relates to the determination of the amylase content of samples by a method that thereby is marked that one

a) to a solution containing a measured amount of the sample, a defined polysaccharide substrate formula

CH ₂ OH

OH

CH ₂ OH

AO J \ 0H / 1_ 0 -f \ OH λ

l / T Υ [/ \ J | / o

OH

CH ₂ OH

where η is an integer in the range from 1 to 10 and R is a substituted aromatic radical,

709886/0633

2 273H21

which, as a separated aglycone, has a different spectral absorbance than the substrate, and

b) monitors the spectral absorbance of the solution.

In the preferred embodiment, η has a value of 2, 3 or 4, and R is a substituted aromatic radical of the formula

x - ^ Jf * kjk ^ ^Or

wherein X and Y individually represent H, N0 ", halogen, alkyl having 1 to 4 carbon atoms, OR ¹ or COpR ¹ , wherein R ^{1 is} an alkyl radical having 1 to 6 carbon atoms, and at least one of the radicals X and Y is an NO ₂ group.

In the preferred embodiments, glycosides of maltotetraose, maltopentaose or glycosides serve as substrates Maltohexaose, in which an aromatic residue is attached to the terminal glycoside unit. With the special preferred embodiment, the terminal glycoside unit is a <x- (4-Nitropheny] ^ glycoside; a maltase is also added to the solution.

The invention further comprises a reagent test pack. This test pack contains one of the above Substrates and a maltase.

The following description of the invention is limited to polymers and oligomers of glucose which

cl / JI > ^ / - are linked t, and in which a substi-

tuierter aromatic residue is bound to the terminal (reducing) glucose unit. These connections have the general formula

709886/0633

X-

CH ₂ OH

II

_ O

in which η is an integer, R is the substituted aromatic radical and the remaining part of the compound of Is glycosyl residue. If it is split off from the glycosyl residue by hydrolysis, the residue R becomes a phenol ROH or an anion R0 ~ of this phenol (depending on the state of the solution), both of which are normally called To be called aglycone.

This compound, which is effective as a substrate for amylase, is a defined polysaccharide or, if η <8, a defined oligosaccharide. The term "defined" as applied to polysaccharides has heretofore been used in the used in a broad sense and often applied to any mixture of polysaccharides in which the relative Quantities of the various polymers and oligomers are known. In the sense used here is the term "defined polysaccharide" is to be understood, however, as a substance which is at least 90% of a polysaccharide with a given chain length, i.e. in which η is a given integer.

α-Amylase acts as a catalyst in the hydrolysis of polysaccharides to short-chain polysaccharides and ultimately to maltose. It has been found, and is reported in the above-mentioned patents, that of all polysaccharides, maltotetraose (G-), maltopentaose (G _1. ) And maltohexaose (G,.) Are preferred for use as a substrate in an amylase determination. The G nomenclature provides a convenient

709 8 8 6/0633

40 273U21

Abbreviation for η <χ / _ϊ > 4 / -linked glucose-

units.

These three substrates are preferred for kinetic and stoichiometric reasons. The binding constant of oc-amylase to polysaccharides is made with

increasing number of α / Τ ^ 47 bonds up to about G ^

bigger where it flattens out. For homologues under G. the binding constant is too small to give acceptable reaction rates. In the case of homologues above G _fi , the reaction is rapid, but the results are not stoichiometric. Unproductive reactions take place so that no n-glucose units are formed when α-amylase reacts with G. Furthermore, the maximum rate at which the substrate is released from the α-amylase becomes lower as the value of η becomes smaller. If glucose is part of the detection system and G is used as substrate, η glucose units should be formed for every interaction of α-amylase with G within a reasonable time. Otherwise, the percentage of the total glucose units released must be estimated in comparison to the units available, and this leads to errors. Because of these factors, G., G_ and G _fi become the preferred substrates.

In the system according to the invention, in which the detection system of the release of a substituted phenol ROH and does not depend on glucose formation, stoichiometric considerations are not very important, and η can range from 1 to 10. However, an oligosaccharide is preferably used as the polysaccharide η in the range from 1 to 8 is used.

As disclosed in U.S. Patent 3,879,263, the use of a maltase such as α-glucosidase is in the measurement neither the α-amylase in the pancreatic secretion nor the total amylase required. This is the case when as

709886/0633

fl 273U21

Substrates the substrates according to the invention as well as G ₄ , G ₅ and G can be used. Furthermore, since the invention is not dependent on the detection of glucose, maltase is not required when using the substrates according to the invention even when determining α-amylase in saliva. However, by using a maltase, the reaction rate is increased under all circumstances. It is especially advantageous to achieve a truly stoichiometric reaction because the rate of reaction of maltase with lower ^ü ligosacchariden is higher than the Geschwindkgkeit the reaction of a-amylase with these substrates. α-Amylase causes the substrate to hydrolyze to smaller fractions and the maltase causes the hydrolysis to be completed to glucose units so that the release of the substituted phenol occurs stoichiometrically. For this reason, oligosaccharides of the above formula, in which η has a value of 2, 3 or 4, are particularly preferred as substrates for the purposes of the invention. The following description is therefore restricted to these substrates, especially those in which η stands for 2 or 3. However, this limitation is only for the sake of simplicity and is not intended to limit the disclosure.

When using a maltase, for example α-glucosidase, a side reaction takes place due to the reactivity of the maltase with the substrate, which leads to a blank rate. This means that phenol is released even in the absence of α-amylase. Since the maximum rate of release of product from maltase becomes lower as the value of η increases, the increase in blind speed is slower than the increase in n. Thus, the blind speed for G- is expected to be lower than that for G ₄ . This is confirmed by tests. However, an additional factor plays a role in choosing between

709886/0633

273U21

higher and lower oligosaccharides (ie, G- or G ', play a role as substrate. In all reactions of the substrate with amylase and all reactions of maltase with the substrate, the rate increases as a function of the substrate concentration to an optimum at which it levels off Substrate concentration at which optimization occurs appears to increase as the value of η increases, so more substrate (and maltase) must be used to optimize (linearize) the standard curve, which is an important consideration for expensive chemicals.

The substrates according to the invention are the defined polysaccharides of the above formula in which η is a integer between 1 and 10 and R is a substituted aromatic radical which, when derived from the polysaccharide in the form of a phenol or phenolate anion

is split off, has a different spectral absorbance than the substrate. There are a large number such remains. However, the main of them are the residues of the formula

and

wherein X and Y individually represent H, NO ₂ , halogen, alkyl having 1 to 4 carbon atoms, OR 'or COpR', wherein R 'is an alkyl radical having 1 to 6 carbon atoms, and at least one of the radicals X and Y is a NOp group. The phenol anions formed by separating these residues from the glycosyl residue have a maximum extinction

X between about 290 and about 600 nm. Max

The details of the methods of making these preferred compounds are set out in the DT-PS (Patent application P of the same day corresponding to US patent application 704 974/5) of the

709886/0633

derin, so that a repetition can be dispensed with here.

Of the preferred embodiments, two compounds are particularly preferred, namely the compounds in which η has the value 2 or 3 and R is a 4-nitrophenyl radical. These compounds, a- (4-nitrophenyl ) maltotetraosid (G.pNp) and oc- (4-nitrophenyl) -maltopentaosicr, are used to determine the amylase content of a sample, e.g. blood serum or urine, in accordance with the following reaction scheme is used:

709 8 8 6/0633

ν -Ak

273U21

CH, OH

HO

OH

OH

CII ₇ OH

OH

CHjOH

-0— · \ OH

R = 4-

λ max 290-305 µm

n = 2a- (4-nitrophenyl) maltotetraoside n == 3 a- (4-nitrophenyl) maltopentaoside

α-amylase

CH ₂ OH

G ₀ or
²

OH

CH ₂ OH

-0-'X OH

CH, OH

R = 4-O ₂ NC ₆ H ₄ n = 0 a- (4-nitrophenyl) maltoside

Maltese

or

4-nitrophenol

OH"

4-nitrophenolate anion λ. 410 nm

The defined oligosaccharide serving as substrate becomes added to a solution containing a measured amount of the sample to be tested, whereupon the spectral absorbance of the solution either as a determination of the end point or as a determination of the speed with the help of conventional methods is monitored. In general, as with all enzyme reactions, the reaction solution at substantially

8 8 6/0633

273U21

constant pH and held at a substantially constant temperature. When using these substances, it is advisable to carry out the determination in a solution whose pH value is set to the basic range in order to increase the extinction at 410 nm. For example, G ₄ pNp and G ₅ pNp (K 290-305 nm) and nitrophenol (^ _x 313 nm) at 410 nm have a low extinction coefficient compared to the 4-nitrophenolate anion (X 410 nm).

In order to best accomplish this, a reagent test kit such as the one noted above is used contains defined substrate and a maltese. Such a test pack is exemplified in US Pat 3 476 515. This test pack can be used in the analyzer described in US Pat. No. 3,770,382 be used.

example 1

A sample of <x- (4-nitrophenyl) maltotetraoside (G.pNp), according to example IH of the DT-PS (patent application

dung P of the same day corresponding to the

US patent application 704 974/5) of the applicant was prepared in 66.7 mmol sodium phosphate buffer of pH 6.5 dissolved in such amounts that various sub-concentrations of 2 to 8 mg / 3 ml are obtained became. As described in the cited patent application, this substrate sample was using a Purified chromatography column from "Sephadex LH-20" been. α-glucosidase in various concentrations from 2.5 to 12.5 international units per 3 ml of substrate solution (IU / 3 ml) was then added to the substrate solution added, the volume of which was brought to 3.0 ml. The solution was incubated at 37 ° C for 1 to 10 minutes.

After measuring the blind speed at 410 nm below

709886/0633

10

273U21

Using a GiIford spectrophotometer was the Reaction by adding 0.1 ml of an "Elevated Enzyme Control Product" (manufactured by the applicant) (1150 Somogyi units per dl (SU / dl) amylase), the 1: 1 was diluted with "Du Pont Enzyme Diluent" (enzyme diluent). This amylase concentration corresponds to approximately six times the upper normal concentration in serum. The overall reaction rate was then measured using the Gilford spectrophotometer. By subtracting the blind speed the pure reaction rate was determined from the overall rate.

A statistical optimization with two variables was made for the substrate and the α-glucosidase. The results of this evaluation are in Table I in arbitrary absorbance units (A) per minute called.

12.5

ro

QJ (0 10 Ό • Η W O

7.5

Table le I O.018 O.005 ¹ O.012 0.110 O.091 ² 0.113 O.092 O. 086 ³ 0. 101 O. Oil 0. 004 0. 008 O.109 0. 090 O.110 O.098 0.086 0.102 0. 002 0. 001 0. 008 O. 080 0.079 O.091 O.078 0.078 O. 08 3

2.5

2.0 5.0 8.0

G ^ pNp mg / 3 ml

1. Blind speed (A / min.)

2. Total speed (A / min.)

3. Pure speed (A / min.)

709 8 8 6/0633

■ ~ ^Y ' 273U21

At

This evaluation shows that the blind speed increases with increasing concentrations of both G.pNp and also the ot-glucosidase increases that the optimal concentration of G.pNp is about 4.0 mg / 3 ml, and that the optimal concentration of α-glucosidase is about 7.5 IU / 3 ml.

Using these optimal values of G.pNp and α-Glucosidase in a reaction solution of 3 ml were the reaction rates for different concentrations the amylase measured in the sample. A standard curve was drawn with the aid of the measured values. the end this curve was the sensitivity in mA / min. / SU / dl measured. The standard curve for this sample is shown in FIG. The blind speed and the Sensitivities for this sample and other examples are given in Table II.

Table II

example Blind speed sensitivity (mA / min. (mA / min. / SU / dl) 1 5.0 0.115 2 not measured 0.231 3 10.3-13.3 0.110 4th 3.0 0.116 Example 2

A small amount of the substrate sample used in the experiment described in Example 1 was passed through High Performance Liquid Chromatography (HPLC), one well known to those skilled in the art Standard cleaning procedure, further cleaned. Using those determined according to Example 1 optimal values for G.pNp and α-glucosidase and the in The amylase sample described in Example 1 established a standard curve using this purified substrate drawn. The sensitivity was as in Deisp. The standard curve is shown in FIG

709886/0633

273H21

shown. The sensitivity values are given in the table Called II. As Table II shows, the sensitivity of the determination became essential by the purification increased, an indication that the substrate sample of Example 1 contained some inhibitor.

Example 3

The α- (4-nitrophenyl) -maltotetraoside used in this experiment (G.pNp) was obtained by deacetylating the HPLC-purified acetate of Example ID of

DT-PS (patent application P of the same day corresponding to US patent application 704 974 / 704 975). Specifically, a solution of sodium methoxide was added to a sample of this acetate and given methanol. The solution was stirred in a closed vessel at room temperature for 18 hours. The methanol was then removed under reduced pressure.

The G.pNp formed in this way was in 66.7 mmol Sodium phosphate buffer of pH 6.5 dissolved in such an amount that a substrate concentration of 4 mg / 3 ml was obtained. Then there was 7.5 IU / 3 ml of α-glucosidase added to the substrate solution. The volume of the solution was brought to 3.0 ml. The solution was 1 to Incubated for 10 minutes at 37 ° C.

After measuring the blind speed in the manner described in Example 1, the reaction was carried out Add 0.1 ml "Du Pont Elevated Enzyme Control Product", which contains the enzyme diluent "Du Pont Enzyme Diluent "in a ratio of 1: 1 was triggered. The reaction rates for different Amylase concentrations in the sample were measured in the manner described in Example 1, followed by a standard curve was drawn. The sensitivity in mA / min. / SU / dl was determined from this curve.

709886/0633

273U21

The standard curve for this substrate is shown in Figure 1, and the blind speed and the Sensitivities are given in Table II.

This is a raw specimen that has not yet been processed by chrotnatographic Separation methods has been cleaned. As a result, the blind speed is very high - it is in the range from 10.3 to 13.3 mA / min. - but the sensitivity is that given in Example 1 Sensitivity of the substrate, in which a previous cleaning with a column of "Sephadex LH-20" had been made, equivalent.

Another series of reactions was carried out under the above conditions, but the reaction 5 min. After its initiation by adding 1.5 ml of an aliquot of the sample solution in either 1.5 ml of o, 2 molar Na ₃ CO ₃ or 5 ml of o, oo2n NaOH was canceled. At pH 6.5, the extinction coefficient of 4-nitrophenol is relatively low because not all of the 4-nitrophenol is ionized. The rise in pH as a consequence of the termination of the reaction is sufficient to completely ionize the 4-nitrophenol to 4-nitrophenylate, which increases the extinction coefficient. This results in an "endpoint" for which the standard curves were not straight, probably because the system was optimized for speed rather than endpoint approximation. However, a five to ten fold increase in sensitivity was observed.

Example 4

One according to the example IG of the DT-PS (patent application P of the same day accordingly of US application 7o4 974/5) prepared sample of <x- (4-nitrophenyl) maltotetraoside was in 66.7 mmol Sodium phosphate buffer of pH 6.5 in such an amount

709886/0633

273H21

dissolved so that a substrate concentration of 4 mg / 3 ml was obtained. Then there was 7.5 IU / 3 ml of α-glucosidase added to the substrate solution. The volume of the solution was brought to 3.0 ml. The solution was 1 to Incubated for 10 minutes at 37 ° C.

After measuring the blind speed in a Gilford spectrophotometer at 410 nm, the reaction was stopped by adding 0.1 ml of "Du Pont Elevated Enzyme Control Product "made with the enzyme diluent" Du Pont Enzyme Diluent "in a ratio of 1: 1 was triggered. The reaction rates for the different Amylase concentrations in the samples were measured on the Gilford spectrophotometer, whereupon a Standard curve was drawn. The sensitivity in mA / min. / SU / dl was measured from this curve. the The standard curve for this sample is shown in FIG. The blind speed and the sensitivity are mentioned in Table II.

This, too, is a substrate that had been purified through a column of "Sphadex LH-20". The sensitivity the determination using the substrate of this example is that of the examples 1 and 3 are equivalent.

However, the blind speed is somewhat slower than that of Example 1 and noticeably slower than that of Example 3.

Example 5 -

A sample of a- (4-nitrophenyl-maltopentaoside (GcpNp), produced according to example 2E of the DT-PS

(Patent application P of the same day corresponding to US patent application 704 974/5), was in 66.7 mmol sodium phosphate buffer of pH 6.5 in such Quantities dissolved so that various substrate concentrations ranging from 4.0 to 12.0 mg / 3 ml were obtained.

α-glucosidase with different activity in the range from 15 to 45 IU / 3 ml was then the substrate

709886/0633

' 273U21

solution added, the volume of which was then brought to 3.0 ml. The solution was 1 to 10 minutes at 37 ° C incubated.

Preliminary tests were carried out using 4.0 mg / 3 ml GcPNp and three α-glucosidase concentrations of 7.0, 14.0 and 28.0 IU / 3 ml. For each of these Concentrations were calculated in the manner described in Example 1, the standard curves using the in Example 1 described amylase sample drawn. In jβίο In the case, the blind speed was 3.0 mA / min. the Standard curves for the three α-glucosidase concentrations are shown in FIG. All curves were not straight, so that determination of the sensitivity was difficult. The sensitivity is however with more than 0.160 mA / min. / ΙΕ / dl estimated. The linearity increased with increasing ok-glucosidase concentration Signs that the α-glucosidase concentration is below the Optimal was.

A two variable optimization was done in the manner described in Example 1. The results this optimization are given in Table III.

709886/0633

273U21

10

15th

20th

ld

α)

(Ω TJ

ω O

45

30th

15th

Tabel III 004 0. 005 O .003 ¹ O. 139 0. 145 O .128 ² 0. 135 0. 140 O 125 ³ 0. 004 0. 012 O. 003 0. 135 0. 133 0. 122 0. 131 0. 121 0. 119 0. 005 0. 007 0. 003 0. 119 0. 114 0. 110 0. 114 0. 107 0. 107 0.

4.0 8.0 12. 0

G ^ pNp mg / 3 ml

speed (A / min.)

2. Total speed (A / min.)

3. Pure speed (A / min.)

This evaluation shows that the blind speed increases only slightly when the G ^ pNp or α-glucosidase concentrations are increased. This is in accordance with the situation with G ₅ explained above. This analysis also shows that the optimal value for G, -pNp or α-glucosidase has not been reached at 12.0 mg / 3 ml or 45 IU / 3 ml. Since G-pNp has a slower or at least stable blind velocity as a function of substrate and cx-glucosidase concentrations, it is preferred as the substrate. However, the high concentrations of Gj-pNp and α-glucosidase required for the determinations limit their preferred status.

709886/0633

Claims (1)

Claims Method for determining the amylase content of samples, characterized in that one a) to a solution containing a measured amount of the sample, a defined polysaccharide substrate formula CH ₂ OH CH ₂ OH OH CH ₂ OH _ 0 in which η is an integer in the range from 1 to 10 and R is a substituted aromatic radical, which, as a separated aglycone, has a different spectral absorbance than the substrate, and b) monitors the spectral absorbance of the solution. 2. The method according to claim 1, characterized in that η is in the range from 1 to 8 and preferably the Has a value of 2, 3 or 4. 3. The method according to claim 1 and 2, characterized in that that R is a substituted aromatic radical of the formula ν X or where X and Y individually represent H, NO ₂ , halogen, alkyl having 1 to 4 carbon atoms, OR ¹ or CO ₂ R ¹ , where R ^{1 is} an alkyl radical having 1 to 6 carbon atoms, and at least one the radicals X and Y is a N0., - group. 709886/0633 INSPECTED 273H21 4. The method according to claim 1 to 3, characterized in that R is a radical of the formula X - is, Process according to Claims 1 to 4, characterized in that a maltase is also added to the solution. Process according to Claim 5, characterized in that a-glucosidase is used as the maltase. Process according to Claims 1 to 6, characterized in that R is a 4-nitrophenyl radical. The method according to claim 1 to 7, characterized in that the solution is substantially keeps constant pH in the basic range and at a substantially constant temperature. Method for determining the amylase concentration of samples, characterized in that a maltase and a substrate made from that of α- (4-nitrophenyl) glycosides of maltotetraose, maltopentaose and maltohexaose existing group to a solution gives a contains the measured amount of the sample, and the change in the spectral absorbance of the solution is monitored, wherein the terminal glycoside unit in the substrate is an α- (4-nitrophenyl) glycoside. Reagent test pack for determining the amylase content of samples, containing a) a defined polysaccharide substrate of the formula CH ₂ OH CH ₂ OH _ 0 _f \ OH 709886/0633 273U21 in which η is an integer in the range from 1 to and R is a substituted aromatic radical, which, when split off from the polysaccharide in the form of a phenolate anion, is another Has spectral absorbance as the substrate, and b) maltase. 11. reagent test pack according to claim 10, characterized in that η is in the range from 1 to 8 and preferably has a value of 2, 3 or 4. 12. reagent test pack according to claim 10 and 11, characterized in that R is a substituted aromatic Remainder of the formula or γ ^ γ where X and Y individually represent H, NO ₂ , halogen, alkyl having 1 to 4 carbon atoms, OR ¹ or COpR ¹ , where R ^{1 is} an alkyl radical having 1 to 6 carbon atoms, and at least one of the radicals X and Y is an N0 group. 13. reagent test pack according to claim 10 to 12, characterized characterized in that it contains α-glucosidase as maltase. 14. reagent test pack according to claim 10 to 13, characterized in that R is a radical of the formula is. 15. Reagent kit according to claim 10 to 12, characterized in that R is a 4-nitrophenyl radical is. 16. Reagent test pack for determining the amylase content in samples, containing a maltase and a substrate from the a- (4-nitrophenyl) glycosides of malto- 709886/0633 273U21 tetraose, Mal topentaose and maltohexaose, the terminal glycoside unit in the substrate being a- (4-nitrophenyl) glycoside. 709886/0633