The isolation and crystallization of the enzyme urease.

Sumner first isolated and crystallized an enzyme (urease) and proved that enzymes are proteins.
One month after publication of the above paper Sumner reinforced his discovery by recrystalizing urease, publishing a follow-up paper:
"Note. The recrystallization of urease," J. Biol. Chem., 70, 1926, 97-98.

In 1946 Sumner was awarded half of the Nobel Prize in Chemistry "for his discovery that enzymes can be crystallized." The other half was awarded to John Howard Northrop and Wendell Meredith Stanley "for their preparation of enzymes and virus proteins in a pure form."

(Thanks to Juan Weiss for the follow-up reference.)