Pauling was thinking about more than hemoglobin. His wide reading had also led him to the subject of protein denaturation.

Even modest heating, mild treatment with acids or alkalis, or simple agitation, like beating egg whites with a fork, could be enough to change a protein's properties and kill its biological activity. This process was called denaturation. Pauling had been especially interested in a recent finding by two Rockefeller Institute researchers that showed that some proteins, if denatured by careful, gentle heating could, if cooled properly, come back to life and regain their original activity. Higher heat, however, denatured them irreversibly. How could that be? To Pauling, it looked like denaturation might be, at least in some cases, a two-step process. The first step was reversible, the second irreversible. Perhaps, he reasoned, that meant that there were two kinds of bonds involved. First-step denaturation might involve relatively weak bonds that were easily broken and reformed; second-step denaturation would involve stronger bonds that, once broken, were impossible to mend.

The stronger bonds, he decided, were likely to be covalent bonds, the sort he had described in detail in some of his papers on the nature of the chemical bond. He had a guess about the weaker bonds, too. He had in his reading some years earlier come across the concept of the hydrogen bond, a link in which a hydrogen atom acted as a weak bridge between two other atoms. He had done some work himself recently on the hydrogen bond in relation to water. How might hydrogen bonds work in proteins?

He thought again about Fischer's polypeptide model, the idea of amino acids linked end-to-end into long chains. He was familiar with a 1931 paper by Hsien Wu, who hypothesized that denaturation resulted from the unfolding of tightly coiled chains. An idea began to take shape, a sort of grand theory of protein formation. To see if his idea was right, Pauling started collaborating with one of the Rockefeller scientists who had done the original work on reversible denaturation: Alfred Mirsky.