L-Aspartic acid L-Asparagine .

Order Information Cat. No.. Content R1 700-101 84 ml 40 ml R2 15 ml R3 17 ml R4 12 ml Pipette into Cuvette Buffer Sample NADH
Water

manuelle Vorschrift

Blank (R1) 1,00 ml

--

Sample 1,00 ml 0,10 ml 0,10 ml 1,40 ml

Note

UV-Test
for the determination of L-aspartic acid and L-asparagine, respectively, in foodstuffs and other materials Principle L-Asparagine is hydrolysed to L-aspartic acid (aspartate) and ammonia by the enzyme L-asparaginase (1). Asparaginase L-Aspartate + NH3 1. L-Asparagine + H20 (1) L-Asparagine + H2O asparaginase, L-aspartate + NH3 In the presence of the enzyme glutamate-oxa(oacetate transaminase (GOT), L-aspartate is converted to oxaloacetate with 2-oxo-glutarate GOT 2. L-Aspartate + KG Oxalacetate + L-Glutamate In the reaction catalyzed by malate dehydrogenase (MDH), oxaloacetate is reduced by reduced nicotinamide-adenine dinucleotide (NADH) to L-malate MDH + + L- Malate + NAD 3. Oxalacetate + NADH + H At first, aspartate is determined according to equations (2) and (3). The amount of NADH oxidized in reaction (3) is stoichiometric with the amount of L-aspartate. Addition of Asparaginase leads to a further decrease of the absorbance of NADH which is stoichiometric with the asparagine concentration (1). The decrease of NADH is determined by means of its absorbance at 334, 340 or 365 nm. Reagents R1 Puffer R2 R3 R4 Standard Phosphate buffer pH 7.2 NADH GOT Asparaginase Concentration Asparagine Concentration Asparagine acid 0,25 mol/l 6 mmol/l 30 U/ml 4 U/ml

Asparagin + Aspartat, at 180 umol/l

(R2)

0,10 ml 1,50 ml

Mix well , after ca. 2 min read absorbances of the solutions (A1). Start reaction by addition of GOT (R3) 0,02 ml 0,02 ml 2,9 kU/l

Mix well; an completion of the reaction (approx. 20-30 min) read absorbances of the solutions (A2). lf the reaction has not stopped after 30 min, continue to read the absorbance at 2 min intervals until the absorbance decreases constantly. Add Asparaginase (R4) 0,01 ml 0,01 ml 1,45 kU/l

mix"; an completion of the reaction (approx. 20-30 min.), read absorbances of the solutions (A3). lf the reaction has not stopped after 30 min, continue to read the absorbances at 5 min intervals until the absorbance decrease is constant lf the absorbance decreases constantly at A3 and A3 resp., extrapolate the absorbances to the time of the addition of GOT (R3) or Asparaginase (R4), respectively. Determine the absorbance differences (A,-A2). Subtract the absorbance difference of the blank AB from the absorbance difference of the sample AS. A L- aspartate = AS - AB Determine the absorbance differences (A2-A3). Subtract the absorbance difference of the blank AB from the absorbance difference of the sample .AS. E Asparagin = ES - EB The absorbance differences measured should as a rule be at least 0.100 absorbance units to achieve sufficiently accurate results (see instructions for performance of assay"). Calculation According to the general formula for calculating the concentration, the equation is: C =
V

Warnings and precautions Do not swallow! Avoiding touch with skin and mucous membranes. Notice the necessary precautions for the use of laboratory reagents. Reagent Preparation The reagent is ready for use Storage and stability Unopened packet components at 2-9 degrees Celsius: until indicated expiration date R1: open in the cooling subject of the device 60 days R2, R3, R4: open in the cooling subject of the device 30 days Materials required in addition Aqua Dest. (Heavy metal and sterile and usual laboratory equipment). Procedure Wavelength: Cuvette : Temperature: Final Volume : 340 nm, Hg 365 nm or Hg 334 nm 1 cm light path 20-25 C 2,74 ml (L-Asparagin acid) 2,75 ml (L-Asparagin) Read against air (without a cuvette in the light path) or against water. Sample solution: 3-65 pg of asparagine + aspartic acid /cuvette (in 0.1-1.5 ml sample volume).

V x

MW

x d x v. x1000

E (g/l )

= final volume [ml]

c = absorption coefflcient of NADH at v = sample volume [ml]

MW = molecular weight of the substance to be assayed ( g/mol ) d

s

= light path [cm]

= Absorption coeffizient von NADH bei Hg 340 nm = 6,3 Hg 365 nm = 3,4 Hg 334 nm = 6,18 ( l x mmol ( l x mmol ( l x mmol
1
1 1

x cm- ) x cm- ) x cm )
-1

lt follows for L-aspartic acid: L- Asparartic acid in g / l sample Solution C=

2,74 x 133,1

x 1 .x 0,1 x 1000

x E=

E 3,647 x

g/l Aspartic acid

mti-diagnostics GmbH ,Limburger Str. 45, D-65510 Idstein ,  ++49(0)6126-9595 262 WEB : www.mti-diagnostics.de eMail : mti@mti-diagnostics.de

 ++49(0)6126-9595 264
Rev. 2008.03

L-Aspartic acid L-Asparagine .

L- Asparagine in g / l sample Solution C=
2,75 x 132,1

manuelle Vorschrift
Literature 5.1. 9ergmeyer, H. U.. Bernt; E., Milering, H. d Pfleiderer, G. (1974) in Methoden der enzymatischen Analyse (Bergmeyer, H. U., Hrsg.), 3. Aufl., 9d. 2 S. 1741-1745, Verlag Chemie, Weinheim. 5.2. Burba, W. 8 Kastning, M. (1971) Stoffwechselphysiologische Untersuchungen an Zuckerrben whrend der Vegetationszeit. I. Glutamin, Glutaminsure, Asparagin und Asparaginsure, Zucker 24, 386. 5.3. Drawert, F., Barton, H. 8 Hagen, W. (1970) Enzymatische Analysenmethoden zur Bestimmung von Wrze und Bierinhaltsstoffen .IV: Bestimmung von L-Glutamat und L-Aspartat und deren Verhalten whrend der Grung und Lagerung bei verschiedenen Bieren, Brauwissenschaft 23, 345-348. 5.4. Greiner, G. 8 Wallrauch, S. (1980) Vergleichende Untersuchungen zur Bestimmung der Asparaginsure und des Asparagin in Fruchtsften und alkoholfreien Erfrischungsgetrnken, Das Erfrischungsgetrnk/ Mineralwasser Zeitung 33, 776-779.

x 1 .x 0,1 x 1000

x E=

E 3,633 x

g/l Asparagin

lf the sample was diluted during preparation, the result must be multiplied by the dilution factor F. Instructions for performance of assay The amount of L-aspartic acid (L-asparagine) in the cuvette should be between 3 pg and 65 Ng. The sample solution must therefore be sufficiently diluted in order to obtain an L-aspartic acid (Lasparagine) concentration between 0.03 and 0.65 g/l. Dilution table estimated amount of LAspartic acid and L-Asparagin per Litre < 0,65 g 0,65 - 6,5 g 6,5 - 65 g Dilution with Water 1+9 1 +99 DilutionFaktor F

10 100 Producing:

If the absorbance difference measured (AA) is too low (e.g. < 0.100), the sample solution should be prepared anew (weigh out more sample or dilute less strongly) or the sample volume to be pipettes into the cuvette can be increased up to 1.5 ml. The volume of water added must then be reduced so as to obtain the sample final volume for the sample and blank in the cuvettes. The new sample volume v must be taken into account in the calculation. 1. Instructions for preparation of sample Filter turbid solutions. Cut solid and semi-solid samples and mix thoroughly (electric mixer, meat grinder or mortar); extract with water or dissolve; Filter, lf necessary. Extract fat-containing samples with warm water, refrigerate for 20 min to separate the fat. Deproteinize protein-containing sample solutions with perchloric acid (1 mol/l) in the ratio 1 + 2 or 3, then centrifuge and neutralize the supernatant with KOH (2 mo/l). Place the solution in the refrigerator for20 min, filter oft the KCIO4 precipitate. Dilute the clear solution, it necessary (see dilution table) and use it for the assay. For the determination of L-aspartate and L-asparagine in sugar beets see reference 3. 2. Specificity L-Asparaginase reacts not only with L-aspartate but also with cystic acid. However, the reaction rate with cystic acid as substrate is very low. Therefore the elimination by means of extrapolation of absorbances (A2 and A3, resp.) is possible. Other compounds do not react. 3. Further applications The method can also be used to analyse pharmaceuticals and in research for the analysis of biological materials. For details of sampling, treatment and stability of the sample see ref. 1 and 2.

Limburger Str. 45 D- 65510 Idstein Phone. 06126-95952 62 Fax. 06126-95952 64

mti-diagnostics GmbH

mti-diagnostics GmbH ,Limburger Str. 45, D-65510 Idstein ,  ++49(0)6126-9595 262 WEB : www.mti-diagnostics.de eMail : mti@mti-diagnostics.de

 ++49(0)6126-9595 264
Rev. 2008.03