Enzyme Kinetics: Measuring Inhibitory Effects and Determining Km and Vmax Values

[Sitix]

Somebody please help me.

Practical:

A calibration graph should be prepared by taking 0.0 (zero, blank), 1.0, 2.0 and 3.0 ml aliquots of an aqueous solution containing both 1.67 mM D-glucose and 1.67 mM D-fructose. Add distilled water to maket it 3 ml. Add 1 ml DNS reagent. Incubate 5 min. Add 3 ml distilled water.Measure with spectrophotometer 540 nm

Do the same with inhibitors

Results:

Standard curve

ml glucose/fructose Absorbance
0,----- 0.0
1,----- 0.69
2,----- 1.44
3,----- 2.15


absorbance
ml sucrose No inhibitor + fructose +urea
0------------0.03------ 0.63------ 0.02
0.25-------- 1.08------ 0.97- ----- 0.43
0.5--------- 1.15------ 1.17- ----- 0.71
1.0--------- 2.29------ 1.71------- 1.02
1.5--------- 2.20------ 2.11------ 1.14


Questions
The standard curve drawn in terms of the molar concentration (in the 3 ml samples; not the volumes of standard mixture) of inverted sucrose (glucose + fructose) in the assay tubes.
From your results, determine:

1) the initial rates of reactions in units of mmole min-1 (ml diluted enzyme)-1
2) the Km and Vmax of the diluted enzyme, using the Lineweaver-Burk plot
3) determine the mode of inhibition for fructose and urea, and their Ki values using the Lineweaver-Burk plot

Please help me

I read around a lot but I just cannot start it

I wanted to use C1v1=C2V2 But I don't have the concentration

Please somebody at least tell me how to start it

 

[epenguin]

Rather sketchily described.

A standard curve is an absorbance plotted against known concentrations of something. When you have a standard curve you can then use it to determine the unknown concentrations e.g. resulting from an enzyme reaction, by measuring absorbance. Your first table looks to be the calibration curve and looks reasonably linear.

Your second table might be the enzyme kinetic experiment, but you do not describe it. Was fructose the inhibitor? Does the third column represent measurement in the presence of an (unstated) constant concentration of fructose? What has urea to do with it?

Probably you have data that tells you the amount of (fructose + glucose) made in a certain time by hydrolysis of sucrose at varying concentrations. You say you don't, you would have to know the concentration of the sucrose solution you started with. You'd know that either from having weighed it out and made up the stock solution, or someone told you. You then know the concentration in the experiments since you pipetted various volumes to make a total volume you should also know.

 

[Sitix]

Thanks for your answer

Sorry panicked a little.

It is an enzyme kinetics practical with inhibitors fructose and urea.

I have the data that tells me the amount fructose/glucose and I put it in my question.

I have figured out a lot by myself but thanks for your answer.