Lecture 15 summary

Proteins. What is a protein? Detection of peptides and proteins. Quantitative analysis of proteins.

We discussed a few features of spectroscopy in general, and atomic absorption spectroscopy in particular. From an analytical standpoint, we are primarily concerned with how these various methods can help us answer the question, "How much?", meaning we would like to know amounts or concentrations of particular substances. Atomic absorption spectroscopy can be a very useful method for determining the concentration of proteins with bound metals. For a single metal-binding protein, its concentration can be very accurately determined, provided the protein is pure enough, the stoichiometry of metal binding is known, and the method of analysis is capable of handling and detecting very small quantities. If the protein is quite pure, this method of determining its concentration serves as an accurate way to determine the molar absorptivity coefficient of the protein at 280 nm. Then we would henceforth have a quick and easy way of measuring a pure protein's concentration by measuring its absorbance at 280 nm.

References for proteins and protein purification :

1. Ninfa and Ballou, Fundamental Laboratory approaches for biochemistry and biotechnology, 1998
2. Boyer, Modern Experimental Biochemistry, 3rd ed., 2000
3. James W Gurd, Practical Approaches to Biochemistry, BGYC23S, 1998/99
4. B. Osterlund and J.-C. Janson, Science Tools from Pharmicia Biotech 2, 3 1997

We had some rather technical material for our reading, and our goal for today and the next lecture is to acquire the necessary background to enhance our understanding of this material. We led off with a brief summary of principles of bioenergetics, and the basic features of photosynthesis. We covered some basic concepts about proteins and peptides and how they relate to our recent discussion of polyprotic acid-base systems. Proteins are polymers of amino acids, and are extremely diverse in their structures and biological functions. We considered the idea of the pI of a protein: Since a protein is typically a very large molecule with very many functional groups that may act as acids and bases, it turns out that a characteristic of a protein is the pH at which the number of positive charges exactly balances the number of negative charges, so that the protein has no net charge (i.e. it is neutral). This characteristic pH value is said to be the pI of the protein. We defined what phosphorylation of proteins entails and the idea that protein phosphorylation changes the charge on a protein, and that it is a regulatory mechanism that controls protein function in various ways. Protein phosphorylation is itself controlled by the competing activities of enzymes called kinases and phosphatases.