The enzymatic activity of the DNase I variants was tested using a DNA hyperchromicity assay. The (spectral) absorbance of a solution of intact DNA was measured at λ = 260 nm. Then the enzyme was added, and the solution was monitored for an increase in absorbance (this is the hyperchromic effect). Why was the hyperchromicity assay effective in assessing the activity of the DNase I variants?
Introduction: The enzyme deoxyribonuclease I (DNase I) is an endonuclease that hydrolyzes the phosphodiester bonds of the double-stranded DNA backbone to yield small oligonucleotide fragments.
DNase I is used therapeutically to treat patients with cystic fibrosis (CF). The DNase I enzyme is inhaled into the lungs where it then acts upon the DNA contained in the viscous sputum secreted by the lungs in these patients. Hydrolysis of high molecular weight DNA to low molecular weight DNA in the sputum decreases its viscosity and improves lung function.
Animal studies have shown that DNase I is also effective in treating the autoimmune disease systemic lupus erythematosus (SLE). In this disease, the DNA secreted into the serum provokes an immune response. DNase I prevents the immune response by degrading the DNA to smaller fragments that are not recognized by the immune system.
Genentech, Inc., the company that produces the recombinant DNase I, was interested in improving the efficiency of DNase I so that less drug would be needed to achieve the same results. Scientists in the protein engineering lab constructed hyperactive variants of DNase I which actually worked better than the wild-type enzyme. DNase I acts by processively nicking the phosphodiester backbone, so the scientists reasoned that a variant that could created more nicks in a shorter period of time would act more efficiently than the wild- type enzyme.
The DNase I variants engineered by the Genentech scientists are listed in the table to the left. Each letter-number-letter combination in the nomenclature of the variant shows the change in an amino acid in the wild-type enzyme. For instance,
“Q9R” means that the glutamine at position 9 in the wild-type DNase I enzyme has been changed to an arginine (find the one-letter amino acid codes).
The enzymatic activity of the DNase I variants was tested using a DNA hyperchromicity assay. The (spectral) absorbance of a solution of intact DNA was measured at λ = 260 nm. Then the enzyme was added, and the solution was monitored for an increase in absorbance (this is the hyperchromic effect). Why was the hyperchromicity assay effective in assessing the activity of the DNase I variants?
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Not just generic "degradation" or even shorter DNA fragments, what specific structure change in double-stranded DNA does the hyperchromicity effect show?
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