Proteins exist to interact with other molecules.  We have substrates converted to products by enzymes, proteins that bind small molecules and proteins that bind other proteins.   Often in the study of a protein system one would like to know the binding constant of a ligand to a receptor or enzyme this is similar to determining a michaelis constant although more specific and not limited to enzymes.  Studies of hormones and antibodies are two of the more notable instances where binding constants are determined.  Binding constants are usually measured using a Scatchard plot where we plot the Bound ligand vs the Bound/Free.  This is a convenient plot for most situations as it identifies both the binding constant and the number of sites ligand can bind to in the system.    If there are multiple binding sites and the binding of the ligand is cooperative then the Hill plot is used as it give both information about the binding constant and information about how the individual sites are interacting.  It is a more difficult plot because more information is needed than in a Scatchard plot.  We will be attempting to do a Scatchard analysis of the binding of NADH to the enzyme Malic dehydrogenase the enzyme that converts malate to oxaloacetate.  .

        Most binding assays are done radioactivity because of the much higher sensitivity possible.  Radioactivity is detectable down to the uMolar level and below.  A Curie of Radioactivity is 3.7x10¹⁰ or so disintegrations per second which is a huge amount of radioactivity, but this only represents 10 fremtomoles of a substance (10^-15 for those of you who don't have that prefix memorized).  Most binding constants are well above this level and most of the time we work with milli or micro Curies of radiation to determine them.    As we currently are not equipt to deal with radiation we will attempt to design an experament based on absorbance or fluorescence to determing a binding constant.   There are some considerations of experimental design for an enzyme assay in Chap 6 .15 in robyt and White.  It would do you well to read that section as it has information pertenent to what we are interested in doing Section 6.14.1 and 6.14.2 have information on quantitating isotope experiments  and Sections 6.1-6.9 are of general interest in understanding the use of radioactivity for use in biochemical studies.
        Section 8.1 and 8.2 in your classroom text talks about the process of binding.