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Determination of the meter constant (Km) and the maximum reaction speed (Vm).

 Last Update: 2020-10-19
 Source: Internet
 Author: User

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acidic phosphatase kinetic properties analysis

m constant (Km) and the maximum reaction speed (Vm

"principle"

under conditions of constant temperature, pH, and enzyme concentration, the substrate concentration has a significant effect on the speed of the enzymatic reaction. When the substrate concentration is very low, the speed (v) of the enzymatic reaction increases rapidly with the increase of the substrate concentration, as the substrate concentration continues to increase, the rate of reaction begins to slow down, and when the substrate concentration increases to a certain extent, the reaction speed reaches a limit value (Vmax).

substrate and the speed of reaction can be represented by the Michaelis-Menten equation:

p style

<" "text-left;" >v- reaction speed;

Km-meter constant;

Vmax-enzyme reaction maximum speed;

?S?

is visible from the Meter equation: the Meter constant Km equals the substrate concentration at half the maximum reaction speed, and the meter constant is in units of concentration (mol/L or mmol/L).

In enzymatic analysis, Km is a fundamental characteristic constant of enzymes, which contains the properties of enzyme-substrate binding and deconsociation. Km is independent of substrate concentration, enzyme concentration, pH, temperature, ion strength and other factors. For each enzymatic reaction, there is a specific Km value under certain conditions and can therefore be used to identify enzymes.

< p style is "text-align: left;" > to determine Km, Vmax, which is generally used as a graph. There are many methods of graphing, the most commonly used is the Linewaver-Burk graphing method, which is based on the countdown form of the Mi equation, in 1/v to 1/S, to obtain a straight line. The straight line has an intercept of -1/Km on the horizontal axis and a long-range of 1/Vmax, and Km and Vmax can be found.

" Methods and steps

< p style-"text-align ">1, make standard curves

takes 9 test tubes, numbered 0 to 8.

tubes 1 to 8 add 0.1 to 0.8mL phenol standard application fluid, and the volume of each tube is replenished with distilled water to 1.0mL, 0 tubes are added to 1.0mL distilled water. Each tube plus 1mol/L sodium carbonate solution 2.0mL and Folin-phenol solution 0.5mL, shake well, 35 degrees C insulation color for 10 minutes. The tube No. 0 is blanked and the absorbent A680 of each tube is read at the visible light10 photonometer680nm wavelength.

the entire operation can be found in the table below:

< p style"text-align: left; "> tube number

"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 7 .

phenol content

< "top" width"41" >

:left;">0.04

0.08

0.12

0.16

0.20

td valign

< "top width" "41" >.

0.28

0.32

0.4mmol/Lphenol standard application liquid/mL

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

distilled water/mL

0.9

. 0.8.

0.7

0.6

0.5

0.4

0.3

0.2

1.0

1mol/L sodium carbonate solution/mL

<p style" text.

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