IB Biology IA, EE, enzymes hydrolysis, glucose assay with colorimeter and Sucrose hydrolysis with sucrase
IA and EE on Glucose Quantification
1. 1% glucose std was prepared ( 1g in 100ml )
2. Perform 2 fold serial dilution ( 1%, 0.5%, 0.25%, 0.125% )
3. Add 2 ml of 1% glucose + 2 ml of DNS (3,5 dinitrosalicylic acid) into a 50ml test tube.
4. Repeat step 1 to 3 with different glucose concentration
5. Place all tubes in boiling water bath for 5 mins for colour formation. ( red brown)
6. Transfer 1 ml of sol from tube to cuvette and added 2ml of water to dilute it
7. Set visible spectrophotometer at 487nm.
8. Prepare a blank containing 1ml DNS + 2ml water.
9. Measure absorbance at 487nm
10. Plot std calibration curve
11. Beer's Law works only for dilute solution.
12. Range for glucose ( 1% to 0.01%)
13. Won't work if too little glucose is used
14. Watch video for clarification
Video, Glucose Quantification with DNS/Visible Spec
Click HERE on glucose quantification and preparation of DNS solution.
* Resulting Glucose/DNS solution must be diluted with water as Beer's Law only applies for diluted solution.
*Experimental Kit can be purchased for EE and IA work. Click HERE to purchase kit
* Glucose sol range must be around 1% to 0.1% for colour determination.
* All item can be purchased from Carolina Biologicals or Kemtecscience
IA/EE, Effect of pH,Temp and Salt on enzyme activity (Invertase/Sucrase)
Break down of sucrose into glucose and fructose measured with DNS/Visible Spec
Introduction on Sucrase and DNS
Glucose( reducing sugar) + DNS form a red brown sol.
Fructose, galactose, maltose and all reducing sugar will react with DNS to form red brown solution
Only sucrose, starch and cellulose (non reducing sugar) will not react with DNS
Investigation on Breakdown of sucrose by sucrase and factors affecting it.
Sucrase break down sucrose into glucose and fructose
Glucose + DNS form red brown solution
Steps to follow
1. Prepare 5% sucrase ( 5g in 100ml buffer pH 7)
2. Prepare 1% sucrose ( 1g in 100ml )
3. Add 2ml sucrose + 10ul enzyme into a test tube
4. Incubate for 5mins at RT
5. Add 2ml DNS to stop reaction/colour formation.
6. Place test tube with DNS into water bath at 95C for 5 mins for colour formation
7. Remove and cool to RT
8. Add 1.5ml water to tube ( diluted as Beer's Law applies )
9. MUST calibrate with a blank ( as step 1 to 8 without adding sucrase)
10. Positive test ( red brown ) presence of glucose/fructose
Effect of pH on Sucrase activity
Expt set up
11. Incubate 1ml enzyme + 1ml pH buffer for 5 mins at RT
12. Add 10ul enzyme/buffer to 2ml sucrose, incubate for 5mins
13. Repeat step 5 to 9 and measure Abs at 487nm
Positive control, add 10ul from a tube containing (1ml 5%enzyme + 1ml water) to 2ml sucrose, then incubate for 5mins
14. Repeat step 5 to 9 and measure Abs at 487nm
Results ( after 300s)
Calc of Glucose from Std Curve ( 300s)
pH 2, Abs = 0.618, Std Curve, glucose is 0.21%
Rate of digestion of enzyme after 300s
= Rate of gluc/fruc produced over 300s
= 7x10^ -4%/s
Click HERE for Data
Assumptions + Tips to make it work
- Sucrose break down to glucose/ fructose and both reducing sugar react with DNS
- Glucose Std Curve is used instead of glucose/fructose Std ( too tedious)
- Time is major factor and error. The longer incubation, more glucose produced. Time must be properly controlled, reaction is time dependent
- Must be diluted with water ( Beer's Law) and it need some trial and error
- Must always calibrate with blank ( see above )
Using this quantification technique on glucose for investigation on
- Hydrolysis of starch by diff amylases (sources) producing glucose
- Factors (pH, ionic salts, temp, inhibitors) affecting enzymatic hydrolysis of starch/sucrose/cellulose using DNS/Visible Spec as quantification method
- Hydrolysis of cellulose with enzyme cellulase ( Biofuel production) producing glucose. Cellulosic ethanol is a biofuel produced from wood, grasses, or the non-edible parts of plts
- Optimization of this technique, more sensitive ( detection below 0.01% glucose ), easy for students to use ( instead of Benedicts and Sugar dip stick)
- Colour stabilisation using potassium sodium tartrate and phenol solution
Click HERE on glucose quantification using DNS
Click HERE on sucrose quantification using DNS
Click HERE for EE Research Questions on enzyme sucrase using DNS
Click HERE to purchase DNS kitClick HERE to view MSDS toxicity of DNS.
Beautiful, simple expt but demand lots of analytical skills. You never know what u can do until u try.You may not know what results come from your actions, but if you do not try, there will be no result, Try it out..........................................Lawrence Kok