The activity determination of hydrogen peroxide enzyme - potassium permanganate titration.

I. Principle
Hydrogen peroxide enzyme belongs to hemoglobin
protease
, contains iron, it can catalytic hydrogen peroxide decomposition into water and molecular oxygen, in the process play the role of transmitting electrons, hydrogen peroxide is both an oxidant and a reducing agent.
R (Fe2 plus) and H2O2⇒ R (Fe3 plus OH-)
. According to R (Fe3-OH-)2-H2O2⇒R(Fe2-) 2-2H2O-O2
, the enzyme's vitality can be determined based on H2O2 consumption or O2 generation. A certain amount of H2O2 solution is added to the reaction system, and the excess H2O2 is titrated with standard potassium permanganate solution (under acidic conditions) after an enzymatic reaction.
the amount of H2O2 consumed can be found by using 5H2O2 plus 2KMnO4 plus 4H2SO4⇒5O2 plus 2KHSO4 plus 8H2O plus 2MnSO4
.
, instruments and appliances
research; 50ml 4 triangular bottles; 10ml acid
titer tube
;
thermostat
water bath pot;
capacity bottles
25ml 1.
,
reagents
10% H2SO4;
0.2mol/L phosphoric acid buffer PH7.8;
0.1mol/L potassium permanganate standard liquid: KMnO4 (AR) 3.1605g, with newly boiled cooling distilled water made into 1000ml, labeled with 0.1mol/L hertic acid solution;
0.1Mol/L H2O2: Commercially available 30% H2O2 approximately equals 17.6Mol/L, take 30% H2O2 solution 5.68ml, dilute to 1000ml, standard 0.1Mol KMn O4 solution (under acidic conditions) calibration;
0.1Mol/L herbic acid: called excellent grade pure H2C2O2.2H2O 12.607g, dissolved with distilled water, fixed capacity to 1L.
fourth, method
. 1. Enzyme extract: take wheat leaves 2.5g to add pH7.8 phosphate buffer a small amount, grind into a homogenous slurry, transfer to a 25 ml capacity bottle, rinse the research with this buffer, and transfer the flushing fluid into the capacity bottle, with the same buffer, 4000r/min centrifugation 15min, the upper liquid is hydrogen peroxide.
2. Take 4 50 ml triangulation bottles (2 assays, 2 controls), measure the bottle to add enzyme solution 2.5 ml, control bottles to add boiled dead enzyme liquid 2.5 ml, and then add 2.5 ml 0.1mol/L H2O2, while timing, in 30 degrees C thermostat water bath insulation 10min, immediately add 10% H2SO4 2.5ml.
3. Titration H2O2 with 0.1mol/L KMnO4 standard solution, ending with pink (not disappearing within 30min).
: Enzyme activity is expressed by the number of milligrams of H2O2 broken down within 1min of each gram of fresh heavy sample:
enzyme activity (Mg/g. Min) (A-B) ×VT×1.7FW×V1×t (3-2)
: A: Titration milliliters (ml) against KMnO4); B: KMnO4 drops after enzyme reaction Fixed number of milliliters (ml); VT: total enzymatic fluid (ml); V1: amount of enzyme fluid used for reaction (ml); W: sample fresh weight (g); 1.7FW: 1 ml 0.1mol/L KMnO4 equivalent to 1.7mgH2O2.
, note that
KMnO4 solution and H2O2 solution used in this process should be recalcuited before use.
.