Characteristics and determination of neuraminidase from Clostridium perfringens in Worthington

Neuraminidase （ Sialidase ） Isolated from a variety of glycoproteins  N- Acetylneuraminic acid （ Sialic acid ）. Neuraminidase is an important tool for studying glycoproteins in protein chemistry and Cell Biology （Hatton etc. , 1973）.

AI Meijie  Worthington  Neuraminidase comes from Clostridium perfringens . stay  Cassidy Based on the work of others , The laboratory developed chromatographic purification methods .（1965  year ）.Wooley  and  Gommi (1966)  The application of this neuraminidase in the method of measuring serotonin receptor is described . It is also used to identify the  N- Acetylneuraminic acid .

 

 

AI Meijie Worthington Characteristics of Clostridium perfringens neuraminidase ：

The best  pH  value ：5.0-5.1 (Burton 1963). stay  pH 4.0  Or above  pH 8.0  There is little or no activity .

Isoelectric point ：5.1（Groome  and  Belyaven 1958）.

Activator ： nothing , Contrary to neuraminidase from Vibrio , The latter requires divalent metals .

Inhibitors ： The use of neuraminidase inhibitors as possible antiviral and antibacterial agents has shown considerable interest .（Khorlin wait forsomeone 1970;Haskell wait forsomeone 1970; and  Tute 1970）.

Stabilizers ： Serum albumin ,0.3 mg/ml (Cassidy et al . 1965).

stability ： The purified preparation is in  4°C  It can be stored stably for at least two years （Cassidy et al . 1965）.

AI Meijie Worthington Determination of neuraminidase ：

Method :  This determination is based on bovine submandibular mucin  (Worthington Code: MU)  Sialic acid released  (NANA)  The measurement of . Under certain conditions , stay  37°C  and  pH 5.0  Under the condition of , One unit is released from bovine submandibular mucin every minute  1  Micromolar sialic acid .Ziegler and Hutchinson (1972) A coupled enzyme assay for the determination of neuraminidase is described .

Reagents ：

9 M  In phosphoric acid  0.2 M  Sodium metaiodate

0.5 M  sodium sulphate

0.755 M  Sodium arsenite is soluble in  0.5 M  Sodium sulfate and  0.1 N H2SO4. By way of  25 gm  Sodium arsenite （ Molecular weight  129.91） Dissolve in  250 ml 0.5 M  Sodium sulfate and add  5 ml 5 N H2SO4  To prepare . To produce a solution , It may need to be heated slightly .

2.5 N  hydrochloric acid

2.5 N HCl  Medium  5%  Phosphotungstic acid

0.6%  Twice crystallized thiobarbituric acid in  0.5 M  Sodium sulfate . By way of  4.5  g  2x  Crystalline thiobarbituric acid is dissolved in  750 ml 0.5 M  From sodium sulfate . Heat to produce a solution and allow it to cool . Will form crystalline precipitates . Use only supernatant for testing .

0.1 M  acetic acid ,pH 5.0

1%  Mucin ,pH 5.0. By way of  100 mgs Worthington  Submandibular mucin （ Code ：MU） Dissolve in  9 ml  Prepare in reagent grade water , Use acetic acid to  pH  Adjust the value to  5.0, And dilute to the final volume  10 ml. The solution is in  pH 5.0  It will appear muddy .

Cyclohexanone . Be careful ： Read the product label for handling instructions .

enzyme ：

Apply the enzyme to  1 mg/ml  Dissolve in reagent grade water . Prepare four dilutions immediately before use , Range from  0.1 mg/ml  To  0.005 mg/ml.

Program ：

Prepare one  37°C  Water bath . Adjust the spectrophotometer to  549 nm. Enter the numbered tube pipette as follows ：

Mucin  0.4  ml

0.1 M  acetic acid  0.5  ml

Every once in a while , Join in  0.1 ml  Corresponding enzyme diluent . Include  0.1 ml  The blank of water replaces the enzyme . stay  37°C  Incubate in a water bath  30  minute . By adding  1 ml 5%  Phosphotungstic acid , Stop reaction regularly . centrifugal  10  minute . Take out each supernatant  0.5 ml  Aliquot the sample and dry clean the test tube . Add  0.1 ml 0.2 M  Sodium metaiodate . Let stand at room temperature  20  minute , Join in  1.0 ml 0.755 M  Sodium arsenite . Shake the tube until the brown disappears , Then join  3.0 ml 0.6%  Thiobarbituric acid . Heat in a boiling water bath  15  minute , Cool in an ice bath  5  minute , Then add  4.6 ml  Cyclohexanone . Extract the color in the cyclohexanone layer by vortex . High speed centrifugation 15 minute .549 Colored cyclohexanone layer and reagent grade water blank .

Be careful ： Final of the sample  A 549 Should not exceed  0.5  Absorption units .