About the ion exchange chromatography - (Aug/29/2008 )

Dear ALL,


I am going to do the experiment in the coming weeks. As I know in lab manual, I need to purify the trypsin from protein sample (trypsin, cytochrome c and bovine serum albumin) with DEAE Sepharose and i need to collect the fractions at FIXED time. After purification, i need to do the trpsin assay by using artificial substrate N-alpa-benzoyl-L-arginine ethyl ester (BAEE)..

since teacher asks me to measure the A280 nm and A 400 nm for the washing fractions and elution. As i know, A 280 nm is used to measure the protein concentration but I don't know the purpose for measuring A400 nm ?

---

Homework question?

---

---

I will not give you the direct answers, but you should be able to figure it out after.

1) think about which approach is easier to conduct
2) think about the initial reaction rate and enzyme to substrate ratio

---

About the question 2, As I know, the initial reaction should be increased ([enzyme] > [substrate]) as the enzyme concentration increased. Over the time passed, the reaction rate should be level off as the substrate conc. is accumulated.

Enzyme activity can be easily determined by graphical means. I need to draw a graph of absorbance vs. time. NEXT I need to determine the best straight line that can be drawn for my data. I may do this by using a linear regression function spreadsheet. After I have drawn the best-fit straight line, enzyme activity can be determined from the slope of the line .

But I think "Fixed time" is able to determine the enzyme acivity. Can anyone tell me what is meaning of :acuurate" and How "acuurate" do i need to determine??

---