During his final years at Caltech, the early 1960s, Pauling started a new line of inquiry with the aid of Emile Zuckerkandl. They proposed an evolutionary theory called the Molecular Clock based on the analysis of hemoglobin from different species. Pauling also used the terms Chemical Paleogenetics and Paleobiochemistry when discussing the Molecular Clock. In their investigations, Zuckerkandl and Pauling compared the amino acid sequences of hemoglobins and speculated how many millions of years ago two species deviated from a common progenitor. In addition to hemoglobin from healthy human adults, they also examined abnormal human hemoglobin.

When Zuckerkandl arrived at Caltech in 1959 as a postdoctoral fellow, Pauling suggested the project to him. Zuckerkandl originally worked with Richard T. Jones, a graduate student at Caltech, who taught Zuckerkandl fingerprinting – the technique he would use to compare the amino acid sequences of various hemoglobins. Fingerprinting is a dual process of paper electrophoresis and paper chromatography, which produces a migration pattern that differentiates between the various amino acids of polypeptide chains. As mentioned in section 25, Vernon M. Ingram devised fingerprinting and successfully analyzed the amino acid difference between normal and sickle cell hemoglobins.

After producing patterns for many species, Pauling and Zuckerkandl compared the fingerprints and concluded which species were closely or distantly related. Thus, they argued that the hemoglobin genes of humans and primates had stabilized before the two organisms diverged evolutionarily. More specifically, they found that there was a closer relationship between the hemoglobin of humans and apes than humans and orangutans. In addition, they stated that human hemoglobin was more similar to pig and cattle than to fish, which substantiated the theory that fish and land animals separated long ago and proceeded to follow different evolutionary paths. Ultimately, they suggested that one amino acid substitution occurs for every eleven to eighteen million years.

Scientists accepted Pauling and Zuckerkandl’s proposal slowly because of the constant rate of evolution that they proposed; however, prominent men of science have noted its impact, and investigators have expanded upon Pauling and Zuckerkandl’s original research.