Specificity of alpha- and beta-Amylases: Alpha-amylase from either saliva or pancreas is an endoglycosidase that hydrolyzes alpha(1,4) linkages internal to long glucan chains. In contrast, beta-amylase from sweet potato is an exoglycosidase that hydrolyzes alpha(1,4) linkages to release maltose from nonreducing ends, but is unable to pass alpha(1,6) branch points, and therefore yields limit dextrins from amylopectin and glycogen. It would be interesting to have students first isolate amylose and amylopectin as described elsewhere, then carry out complete hydrolysis of both polysaccharides with both amylases and analyze the products by gel filtration chromatography. There would be many lessons in such an exercise: separation of polysaccharides, isolation of and characterization of catalytic differences between endo- and exoglycosidases, and gel filtration chromatography.

Kinetics of Alkaline Phosphatase: A continuous assay for alkaline phosphatase uses p-nitrophenyl phosphate as substrate. p-Nitrophenol release can be followed by absorption at 410 nm. I want to try making what would be the perfect competitive inhibitor for the reaction, p-nitrobenzyl phosphonate, whose C-P bond the enzyme couldn't hydrolyze, but which should be essentially isosteric with the substrate. I'm pretty confident this inhibitor could be easily prepared by treating p-nitrobenzyl bromide or chloride (commonly-used carboxylic acid derivatizing agents) with triethyl phosphite, which would nucleophilically displace the halide. Acid or base hydrolysis of the ethyl esters would yield the product. If a noncompetitive inhibitor of the enzyme is available (?), this would be a perfect kinetics exercise.

Protein Denaturation: Denaturation of a protein such as bovine serum albumin by, for example, pH extremes or urea, would be probed by fluorescence, polarimetry (if you have a polarimeter), and/or viscosity (which can be measured with simple devices such as a capillary micropipet attached to the end of a Pasteur pipet, observing changes in drain times).

Revised 6/14/07