Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
  • G01N33/14—Beverages
  • G01N33/143—Beverages containing sugar
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/823—Acetobacter

Definitions

• This invention relates to a method and agent for use in the enzymatic determination of glucose.
• Analytical methods involving enzymes are, in general, characterized by their high substrate specificity and allow for the exact determination of the substrate in the presence of most accompanying materials which are characterized by similar constitution and which represent disturbing fac tors in the same determination using non-enzymatic analytical methods.
• Enzymatic methods for the determination of glucose have also already been proposed and introduced into practice and include, for example, the reaction of glucose with glucose oxidase yielding D-gluconic acid and hydrogen peroxide and the reaction with hexokinase and adenosine triphosphate producing glucose-6-phosphate.
• the reaction products produced, i.e., hydrogen peroxide and glucose-6- phosphate, thereupon are converted in a further reaction into products which can be measured colorimetrically or photometrically (of M. W. Slein in H. U. Bergmeyers Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, page 117; and H. U. Bergmeyer and E. Bernt, loc. cit., page 123).
• phosphate donor for the new enzyme there can, according to the invention, be used not only the acetyl phosphate mentioned by Kamel et al. (supra) but also all phosphates having the following formula:
• R is an organic radical, which does not possess a negative charge in the immediate vicinity of the phosphate group.
• the new enzyme is, therefore, best designated acyl phosphate :glucose-6-phosphotransferase.
• the enzyme i.e., acyl phosphate:glucose transferase preferentially reacts with those phosphate donors in which the eifect of a negative charge which may be present on the bond phosphate donor-enzyme is the smallest; see, for example, glutaroyl phosphate, succinyl phosphate and 3 phosphoglyceric acid-l-phosphate in comparison with benzoyl phosphate.
• benzoyl phosphate is employed as phosphate donor, the enzyme reacts more rapidly than it does with acetyl phosphate.
• R is an organic radical, which radical does not possess a negative charge in the immediate vicinity of the phosphate group, and, as transferase, there is used acyl phosphate glucose-6-phosphotransferase.
• the course of the reaction in the process according acyl phosphate glucoseG-phosphotransferese (triphnsphopyridinenucleotide) (fi-phosphogluconic acid) 6PG TPNH H+
• the new agents according to the present invention for use in the determination of glucose comprise a phosphate donor, a transferase, glucose-6-phosphate-dehydrogenase and triphosphopyridine-nucleotide and, advantageously, a buffer.
• the phosphate donor forming a component of the new agent is a phosphate having the Formula I and the transferase is acyl phosphate:glucose-6-phosphotransferase.
• the sample to be investigated is mixed with an acyl phosphate having the Formula I, triphosphopyridinenucleotide (TPN), a buffer (as for example, glycocoll buffer, triethanolamin buffer and tris-buffer) and glucose- 6-phosphate-dehydrogenase.
• TPN triphosphopyridinenucleotide
• a buffer as for example, glycocoll buffer, triethanolamin buffer and tris-buffer
• glucose- 6-phosphate-dehydrogenase glucose- 6-phosphate-dehydrogenase.
• Any glucose 6 phosphate which may be present will now react and can be determined beforehand.
• the acyl phosphate:glucose-6-phosphotransferase is then added and, after some minutes, the glucose is determined photometrically on the basis of the reduced triphosphopyridine nucleotide (TPNH) formed in the ensuing reaction.
• TPNH triphosphopyridine nucleotide
• the preferred range of pH is between 8 and 9.5 and
• acyl phosphates of Formula I which are, after all, mixed acid anhydrides, can only be stored for a limited period of time
• the acyl phosphate required for use in a routine procedure can advantageously be synthesized immediately before theglucose determination in a preceding enzymatic reaction, preferably in the cuvette being used for the photometric determination.
• acetyl phosphate can be prepared from a stable acetate and ATP utilizing acetate-kinase and carbamyl phosphate can be prepared from carbamic acid and ATP using therefor carbamate-phosphokinase.
• An especial advantage of the process according to the present invention in the enzymatic determination of glucose, in comparison with the previously known processes, is the high substrate specificity of the combination of acyl phosphate: glucose 6 phosphotransferase with glucose-6-phosphate-dehydrogenase.
• Reagents End concentration 2.50 ml. of glycocoll bulfer having a pH of 9.0 (200 mM.) mM 0.10 ml. TPN (11 mM.; 10 mg./ml.) mM 0.37 0.10 ml. carbamyl phosphate (140 mM.; 20
• EXAMPLE 3 Glucose determination with nicotoyl phosphate The measurement procedure was carried out as in Example 1 but using nicotoyl phosphate (end molarity 3.3 mM.) in place of the carbamyl phosphate. In this case, a 0.01 M a-D-glucose solution was prepared and analyzed. In the glucose determination, there were used 0.02
• acyl phosphate Mg percent glucose-6ph0sphowith HK/ Deviation, transferase G G-PDH percent EXAMPLE 6
• fructose was determined by the method of H. Klotzsch and H. U. Bergmeyer (cf. H. U. Bergmcycr, Methoden der enzymatischen Analyse,
• R is an organic radical which does not possess a negative charge in the vicinity of the phosphate group and acyl phosphate:glucose-6-phosphotransferase, thereafter reacting the glucose-6-phosphate formed with triphosphopyridinenucleotide (TPN) and glucoSe-6-phosphate-dehydrogenase to form reduced triphosphopyridine nucleotide and spectrophotometrically determining the glucose present on the basis of the reduced triphosphopyridine-nucleotide.
• TPN triphosphopyridinenucleotide
• glucoSe-6-phosphate-dehydrogenase glucoSe-6-phosphate-dehydrogenase
• said phosphate donor is a member selected from the group consisting of benzoyl phosphate, carbamyl phosphate, nicotoyl phosphate, acetyl phosphate, propionyl phosphate, and N- benzoyl-glycol phosphate.
• An agent for use in the enzymatic determination of glucose in a sample containing glucose and fructose which comprises a phosphor donor selected from the group consisting of nicotoyl phosphate and benzoyl phosphate; acyl phosphate:glucose-6-phosphotransferase; glucose-6-phosphate-dehydrogenase; and triphosphopyridine-nucleotide.
• Agent according to claim 7 additionally containing a buffer.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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• 1951
  • 1951-01-28 DE DE19511598067 patent/DE1598067A1/de active Granted
• 1966
  • 1966-08-10 US US571416A patent/US3509025A/en not_active Expired - Lifetime
  • 1966-08-15 CH CH1171766A patent/CH488186A/de not_active IP Right Cessation
  • 1966-08-15 GB GB36385/66A patent/GB1108770A/en not_active Expired
  • 1966-08-15 NO NO164305A patent/NO117818B/no unknown
  • 1966-08-16 JP JP41053872A patent/JPS503674B1/ja active Pending
  • 1966-08-16 BE BE685538D patent/BE685538A/xx unknown
  • 1966-08-16 SE SE11081/66A patent/SE337492B/xx unknown
  • 1966-08-17 NL NL6611550A patent/NL6611550A/xx unknown

Non-Patent Citations (1)

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