SUMMARY

• 5.1 The Proteome Is the Functional Representation of the Genome

  The rapid progress in gene sequencing has advanced another goal of biochemistry—the elucidation of the proteome. The proteome is the complete set of proteins expressed and includes information about how they are modified, how they function, and how they interact with other molecules. Unlike the genome, the proteome is not static and varies with cell type, developmental stage, and environmental conditions.

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• 5.2 The Purification of Proteins Is the First Step in Understanding Their Function

  Proteins can be separated from one another and from other molecules on the basis of such characteristics as solubility, size, charge, and binding affinity. SDS–PAGE separates the polypeptide chains of proteins under denaturing conditions largely according to mass. Proteins can also be separated electrophoretically on the basis of net charge by isoelectric focusing in a pH gradient.

• 5.3 Immunological Techniques Are Used to Purify and Characterize Proteins

  Proteins can be detected and quantified by highly specific antibodies; monoclonal antibodies are especially useful because they are homogeneous. Monoclonal antibodies to the estrogen receptor can be generated and then used as a tool to purify the receptor with the use of the technique of immunoprecipitation. Enzyme-linked immunosorbent assays and western blots of SDS–polyacrylamide gels are used extensively.

• 5.4 Determination of Primary Structure Facilitates an Understanding of Protein Function

  The amino acid composition of a protein can be ascertained by hydrolyzing the protein into its constituent amino acids. The amino acids can be separated by ion-exchange chromatography and quantitated by their reaction with fluorescamine. Amino acid sequences can be determined by Edman degradation, which removes one amino acid at a time from the amino end of a peptide. Longer polypeptide chains are broken into shorter ones for analysis by specifically cleaving them with a reagent that breaks the peptide at specific sites. Mass spectrometry allows determination of protein mass, identity, and primary structure. Amino acid sequences are rich in information concerning the kinship of proteins, their evolutionary relationships, and diseases produced by mutations. Knowledge of a sequence provides valuable clues to conformation and function.