Chapter $4/$ Enzyme Purification $119$

If bovine LDH is known to have a temperature optimum around $37C,$ why is it important to purify it at $4C?$

Explain why you should not lose any LDH activity when you centrifuge a crude sample at $20,000g.$

Explain why two different percent saturation levels of ammonium sulfate are used in these experiments.

Explain the physical interactions of the molecules that lead to proteins falling out of solution in high salt. What is the driving force of protein precipitation?

Explain why it is important to add ammonium sulfate slowly.

An enzyme$-$catalyzed reaction produces a product that has a maximum absorbance at $412nm.$ The extinction coefficient is $4000M^{-1}c{m}^{-1}.$ A $10-$to$-1$ dilution of the enzyme is made and $20L$ of the dilution are put into a cuvette with $80L$ of water and $1\mathrm{.}9mL$ of reaction cocktail. The absorbance change per minute is $0\mathrm{.}05.$

a$.$ How many units are in the cuvette if $1U=1mo\frac{l}{m}in?$

b$.$ How many units are in the cuvette if $1U=1$nmo$\frac{l}{m}in?$

c$.$ What is the relative activity of the undiluted enzyme $1mo\frac{l}{m}in$