They were shrouded in a certain mystery, these biomolecules called proteolytic enzymes. We know them today as the protein catalysts that, for example, digest proteins in the foods we eat. These were large proteins, that much was known in the 1930s; but they were of unknown composition and structure, and they could not be differentiated from one another chemically.
That mystery created a certain fascination for Hans Neurath, a young chemist fresh out of graduate school. Ultimately, as founding chairman and professor of biochemistry at the UW School of Medicine, Neurath and colleagues would solve the puzzle of many protein structuresthe proteolytic enzyme, trypsinogen, for exampleand would lay the foundation for a great many other discoveries, including later work at the UW by Earl Davie to understand the blood coagulation process.
By 1939, other researchers had succeeded in isolating and crystallizing a variety of proteins: pepsin, pepsinogen, chymotrypsinogen, trypsinogen, trypsin, carboxypeptidase, and others. Not only were these biomolecules difficult to obtain from animal tissues, but also the techniques available in those days to isolate, crystallize, and measure the degree of purity of these proteins were very crude.
Neurath recalls standing in the cold room of the anatomy department at Duke University, using a band saw to make slices from 100-pound lots of frozen cow pancreas glands. From the tissue fluids, they isolated, by means of "tedious operations," samples of the enzyme carboxypeptidase. In another episode, Neurath recalls driving a truck, loaded with vats of sulfuric acid, to a slaughterhouse to collect fresh cow pancreas glands from which he prepared about 1 gram each of trypsin and chymotrypsinogen.
"The thrill of seeing enzyme crystals was no less at that time than it is today," Neurath reflects, noting in contrast that nowadays, when x-ray crystallographers crystallize the protein of their choice, they are able to derive and visualize its 3-dimensional structure at the atomic level.
In 1950, Neurath joined the faculty of the University of Washington, where Earl Davie was his graduate student; he was later joined by postdoctoral fellow Ken Walsh, who today holds the Earl W. Davie/ZymoGenetics Chair of Biochemistry at the UW.
Neurath describes those heady times as the golden years of protein researchand of government support. "Protein chemistry was clearly in the forefront of biochemical research" at that time, he notes. Using newly developed technologies for determining the sequence of amino acids in proteins, Neurath and colleagues first elucidated the sequences of key portions of several proteolytic enzymes and then decided to "go the whole way" and determine the complete amino acid sequence of trypsinogen, described in a landmark paper published by Walsh and Neurath in 1964.
These early studies have been a watershed for many subsequent developments. Neurath and collegues established experimental proof for the concept of structural similarities among a series of proteases, and gave rise to the concept of protease families. Furthermore, analyzing the protein sequences showed that the blood protein thrombin, a blood-clotting enzyme, resembles trypsin and that this trypsin-like structure is involved in the active portion of the blood coagulation proteases.
Later, Neurath and colleagues also determined the complete amino acid sequence of carboxypeptidase A and the nature of the process that activates this protein-digesting enzyme of the small intestine. Carboxypeptidase was the first protease known to contain an atom of zinc as part of the "active site" of the enzymethe reactive heart of the molecule that is responsible for its special catalytic activity. Besides elucidating the 3-dimensional structure of the enzyme, researchers were able to determine the nature and location of the zinc atom, and the arrangement of atoms around it, under various experimental conditions. The discovery stands as an important milestone on the road to understanding the relationship between molecular structure and the biological function of proteases and other enzymes. Neurath's research has been supported continually for 46 years by the National Institutes of Health, whose records list him among the earliest and longest recipients of awards in its research grants program.