This question is regarding the paper at this link it is free

This question is regarding the paper at this link: (it is free!) https://www.ncbi.nlm.nih.gov/pubmed/9565591

Question 2:     a) To measure the activity of the mutants the researchers used a DNA-Methyl Green Assay. How does this assay work? What would they see if a mutant had increased activity?

b) Which mutants had the greatest activity?

c) Why can the hyperchromic effect be used to test for DNase I activity?

d) Describe how the increasing mutations effected the apparent K_mand V_maxvalues.

Solution

a) To measure the activity of the mutants the researchers used a DNA-Methyl Green Assay. How does this assay work? What would they see if a mutant had increased activity?

Answer: The methyl green assay was implemented to measure hydrolyctic activity of DNAase I on DNA in the presence of divalent magnesium & calcium. This mixture have to be mixed with methyl green to produce the DNA-methyl green substrate. The enzyme-linked immunosorbent assay has used to examine these activities by double antibody labeling. Therefore, hyperactive variants & the activity of DNA hyperactive DNA mutants can be analyzed finally concluded that single basic residue replacements with higher activity compared to wild-type ones.

b) Which mutants had the greatest activity?

The hyperactivity variants or mutants with positive charge have several-fold activity compared to wild type due to procesisve nicking

Ques-c:

The hyperchromic effect be used to test for DNAase I activity because the polymeric substrate nature of DNA at A ₂₆₀, that cause DNA absorption, followed by higher function of degradation by DNAase therefore absorbance signal varied