Anfinsen's success with protein folding inspired him to publish The Molecular Basis of Evolution in 1959. This ambitious book attempted to show the scientific and disciplinary affinities between molecular genetics and protein chemistry. Anfinsen believed that the mystique surrounding deoxyribonucleic acid (DNA), the structure of which was described by Francis H. C. Crick and James D. Watson, was overshadowing the field of protein chemistry. He was dismayed that his work in the late 1950s with bovine pancreatic ribonuclease (RNase) did not hold the same cachet as the well-publicized quest among molecular biologists and geneticists who were seeking to "crack the code" of DNA.
Anfinsen had even more reason to believe that protein chemistry was not accorded the scientific attention it deserved after the worldwide publicity that followed the 1961 announcement that two fellow NIH researchers, Marshall Nirenberg and Heinrich J. Matthaei, had deciphered the first word of the genetic code. Still, Michael Young, one of Anfinsen's many postdoctoral students, believed that "several individuals were stimulated to work in the Anfinsen laboratory largely because they had read this small but scientifically rich publication." (Viewers can read the entire text of The Molecular Basis of Evolution in the Documents section.) Anfinsen returned to this theme 25 years later when he implored geneticists to re-examine "Classical Protein Chemistry in a World of Slicing and Splicing," the title of a paper he gave at the State University of New York at Stony Brook in May 1984.
Beginning in 1963, Anfinsen shifted his research away from bovine pancreatic RNase and began investigating the bacterium Staphylococcus aureus. By 1966, Anfinsen and his colleagues had isolated the Staphylococcus aureus RNase through the use of affinity chromatography, an innovative laboratory technique first developed in 1951 by Dan Hampston Campbell, a professor of immunology at the California Institute of Technology. Affinity chromatography enabled Anfinsen to put a bacterium into a chemical solution that selectively captures molecular particles and spreads them out across a medium so that they can be easily examined, in analogy to the colors of a spectrum. Through this process, Anfinsen could analyze the fragments of the three-dimensional amino acid chain. In all of his experiments, the fragmented pieces of the chain refolded into the native conformation. In 1966 and 1967, Anfinsen took the study of Staphylococcus RNase a step further. Using this technique, he was able to show the complete sequence of the 149 units in the enzyme's amino acids chain. This meant that Anfinsen and his lab researchers could pinpoint the exact positions of the individual amino acids and the peptide bonds through which they are linked.
The significance of this work was to demonstrate that elucidating the chemistry of proteins was essential to understanding the function of RNA in heredity. In 1968, Anfinsen proposed to investigate the way that protein structure created biological activity by artificially modifying enzymes and then seeing how their function was affected. Describing this work in an interview with Israel's Jerusalem Post in January 1970, Anfinsen stated that "[W]e are engaged in what you may call molecular engineering. We look at the structure of an enzyme, for instance, and if we see a loop in the chain that doesn't seem to be doing anything, we see what happens if we chop it off. Some people seem to think that the most important discoveries have already been made in molecular biology, that the double helix wrapped it up, but I think that [protein folding] just opened up an enormous field for us."
Anfinsen's vital contribution to understanding the function and structure of protein folding and conformation in the RNases of bovine pancreas cells and Staphylococcus aureus was complemented by the contemporary discoveries of Stanford Moore and William H. Stein, senior biochemists at the Rockefeller University in New York City. In December 1972, the Nobel Foundation awarded all three scientists the Nobel Prize in Chemistry.