Action Pattern of Human Pancreatic and Salivary α-Amylase on 1,4-α-<i>D</i>-Nitrophenylmaltooligosaccharides. <i>1,4-α-D-Nitrophenylmaltooligosaccharides as Substrates of α-Amylase, I</i>

Wallenfels, Kurt; Laule, Gerhard; Meltzer, B

doi:10.1515/cclm.1982.20.8.581

Cited by 3 publications

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References 2 publications

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“…Since the lag phase of the reaction sequence releasing 4-nitrophenol largely depends on the concentration of α-glucosidase within the assay System (32,33,34), we tried to abridge this phase by addition of aglucosidase from yeast. However, neither the lag phase nor the net absorbance increase in the linear portion of the activity curve were influenced by this enzyme up to 250 k U/l within the assay mixture, thus confirming the observations of David (32) and Rauscher et aL (17) who used 4-nitrophenyl glucosides for the measurement of α-amylase activity.…”

Section: Interferencesmentioning

confidence: 99%

“…The cleavage by -glucosidase of glucosides and 4-nitrophenyl glucosides increases with decreasing chain length of the Substrate (32,33). Since a-glucosidase degrades the original Substrates to smaller ones which are then hydrolysed more räpidiy, the blank reaction accelerates with time after mixing Substrate with auxiliary enzyme (tab.…”

Section: Stability Of Reagents and Intrinsic Sensitivitymentioning

confidence: 99%

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Evaluation of α-Amylase Assays with 4-Nitrophenyl-α-oligosaccharides as Substrates

Lorentz¹

1983

Clinical Chemistry and Laboratory Medicine

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Measurements of -amylase with 4-nitrophenyl glucosides offer the following advantages over methods that rely on the formation of NADH: a short lag phase, no apparent interference by metabolites and enzymes of the sample and extremely stable Substrates with low blank values. The intrinsic sensitivity of nitrophenyl formation was equal to that of hydrolysis of maltotetraose, but was less than that of glucose-producing methods using oligosaccharides. In contrast to starch, the chromogenic Substrates are more rapidly hydrolysed by salivary than by pancreatic amylase.Disadvantages of these Substrates are:higher turnover by animal than by human amylases, and a marked susceptibility of the chromophore to small changes of pH and protein concentration. Some analytical qualities such äs specificity, accuracy, precision, stability of the Substrate and linear ränge are described in detail and compared with those of other methods. Bewertung von Methoden zur Bestimmung von a-Amylase mit 4-Nitrophenyl-a-oligosacchariden als SubstratenZusammenfassung: -Amylase-Bestimmungen mit 4-NitrophenyIglucosiden sind Methoden, die auf einer Reduktion von NAD beruhen, durch eine kürzere lag phase, fehlende Störung durch Metaboliten und Enzyme der Probe und durch außerordentlich haltbare Substrate mit niedriger Leerreaktion überlegen. Die Empfindlichkeit der Nitrophenolbildung entspricht der Spaltung von Maltotetraose, sie bleibt jedoch hinter dem Entstehen von Glucose aus Oligosacchariden zurück. Im Gegensatz zu Stärke werden die chromogenen Substrate schneller durch Speichel-als durch Pankreasamylase abgebaut.Nachteile ihrer Verwendung sind ein höherer Umsatz durch Amylasen tierischer Herkunft im Vergleich zu menschlichen Enzymen und der starke Einfluß geringer Veränderungen der Proteinkonzentration und des pHWertes im Ansatz. Im einzelnen werden die Spezifität, Richtigkeit und Präzision dieser Verfahren, die Haltbarkeit ihrer Substrate, der lineare Meßbereich und ein Vergleich mit anderen Methoden beschrieben.

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Section: Interferencesmentioning

confidence: 99%

Section: Stability Of Reagents and Intrinsic Sensitivitymentioning

confidence: 99%

Evaluation of α-Amylase Assays with 4-Nitrophenyl-α-oligosaccharides as Substrates

Lorentz¹

1983

Clinical Chemistry and Laboratory Medicine

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“…Conventional methods for measuring amylase activity include monitoring the degradation of chromogenic substrates (Filova et al ., ) such as blue starch (Miwa, ), β ‐limit dextrin (Kruger and Marchylo, ) or p ‐nitrophenyl derivatives of oligosaccharides (Wallenfels et al ., ; Moneghini et al ., ; Tokutake et al ., ). These methods, which are commonly used for biological samples (e.g.…”

Section: Introductionmentioning

confidence: 98%

An analytical method for measuring α‐amylase activity in starch‐containing foods

Koyama

Hirao²,

Toriba

et al. 2012

Biomedical Chromatography

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The quality of starch-containing foods may be significantly impaired by contamination with very small amounts of α-amylase, which can enzymatically hydrolyze the starch and cause viscosity loss. Thus, for quality control, it is necessary to have an analytical method that can measure low amylase activity. We developed a sensitive analytical method for measuring the activity of α-amylase (from Bacillus subtilis) in starch-containing foods. The method consists of six steps: (1) crude extraction of α-amylase by centrifugation and filtration; (2) α-amylase purification by desalting and anion-exchange chromatography; (3) reaction of the purified amylase with boron-dipyrromethene (BODIPY)-labeled substrate, which releases a fluorescent fragment upon digestion of the substrate, thus avoiding interference from starch derivatives in the sample; (4) stopping the reaction with acetonitrile; (5) reversed-phase solid-phase extraction of the fluorescent substrate to remove contaminating dye and impurities; and (6) separation and measurement of BODIPY fluorescence by HPLC. The proposed method could quantify α-amylase activities as low as 10 mU/mL, which is enough to reduce the viscosity of starch-containing foods.

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2-Chloro-4-nitrophenyl-ß-D-maltoheptaoside: A New Substrate for the Determination of α-Amylase in Serum and Urine

Henkel¹,

Morich²,

Henkel³

1984

Clinical Chemistry and Laboratory Medicine

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Summary:Tests for the determination of -amylase activity based on oligosaccharides or their 4-nitrophenyl derivatives äs Substrates suffer from some disadvantages. Using a more acid indicator molecule and connecting this group in the ß-form to maltoheptaose leads to a new Substrate, 2-chloro-4-nitrophenyl-ß-D-maltoheptaoside, for the determination of -amylase activity.Due to its pK value, only half of the 4-nitrophenol formed from 4-nitrophenyl-a-D-maltoheptaoside is spectrophotometrically detectable, whereas the total product from the new Substrate is responsible for a measurable absorbance. In addition the molar lineic absorbance of the product is 1.8 times higher. Thus the new test is more sensitive than those employing 4-nitrophenyl derivatives.Both P-type and S-type -amylase isoenzymes have the same affinity for the new Substrate; this is not the case for all the other Substrates, i.e. oligosaccharides and their 4-nitrophenyl derivatives, used in -amylase tests.The sample volume may be reduced, thereby removing interference by substances like bilirubin, glucose and haemoglobin.The test is insensitive to changes in temperature and pH and can be adapted to all mechanized analytical instruments. 2-Chlor-4-nitrophenyl~ß*D-maltoheptQOsid: Ein neues Substrat zur Bestimmung von a-Amylase in Serum und HarnZusammenfassung: Die Verfahren zur Bestimmung von -Amylase auf der Basis von Oligosacchariden und den 4-Nitrophenolderivaten unterliegen einer Reihe von Störfaktoren, die zum Teil erhebliche praktische Bedeutung haben. Durch Einführung einer acideren chromophoren Gruppe und deren Kopplung in ß-Form wird als neues Substrat für die -Amylasebestimmung 2-Chlor-4-nitrophenyl-ß-D-maltoheptaosid vorgestellt.Aufgrund der höheren molaren linearen Absorbanz und aufgrund eines von den bisherigen Tests abweichenden Spaltmusters der Amylase weist der neue Test eine höhere Empfindlichkeit auf.Auch die unterschiedliche Substrataffinität der Amylase-Isoenzyme, die von den bisher gebräuchlichen Tests bekannt ist, spielt bei dem neuen Substrat eine untergeordnete Rolle.Das Probenvolumen kann reduziert werden und daraus resultiert eine geringere Empfindlichkeit gegenüber den Störfaktoren der Probe wie Bilirubin, Glucose und Hämoglobin.Besonders unempfindlich ist das neue Testsystem gegenüber Schwankungen im pH-und Temperaturbereich.

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Action Pattern of Human Pancreatic and Salivary α-Amylase on 1,4-α-D-Nitrophenylmaltooligosaccharides. 1,4-α-D-Nitrophenylmaltooligosaccharides as Substrates of α-Amylase, I

Cited by 3 publications

References 2 publications

Evaluation of α-Amylase Assays with 4-Nitrophenyl-α-oligosaccharides as Substrates

Evaluation of α-Amylase Assays with 4-Nitrophenyl-α-oligosaccharides as Substrates

An analytical method for measuring α‐amylase activity in starch‐containing foods

2-Chloro-4-nitrophenyl-ß-D-maltoheptaoside: A New Substrate for the Determination of α-Amylase in Serum and Urine

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