In the 1930s, whether enzymes were proteins and what viruses were made of were major unanswered questions. Northrop extracted pepsin and trypsin from bovine stomachs and achieved high-purity crystals using pH control, dialysis, and salting-out. Stanley purified plant sap containing tobacco mosaic virus and crystallized it, showing that viruses are proteinaceous particles. These findings confirmed that biocatalysts and pathogens share a common chemical basis. The techniques have been applied to screen enzyme inhibitors and to produce large amounts of vaccine antigens, giving them high societal value. Having pure crystals greatly improved the accuracy of X-ray structure analysis and immunological tests.

Northrop precisely controlled ionic strength and isoelectric points in buffered solutions, crystallizing pepsin (pI ≈ 1.0) and trypsin (pI ≈ 10.5) and determining their specific activities and molecular weights. He employed sedimentation velocity and freezing point depression to estimate molecular masses, providing decisive evidence that enzymes are proteins. Stanley purified tobacco mosaic virus using ammonium sulfate precipitation and pH adjustment, obtaining rod-shaped crystals. Subsequent X-ray diffraction revealed TMV to be a helical protein assembly about 300 nm long and 18 nm in diameter. These isolation and crystallization methods stimulated the development of column chromatography and influenced vaccine bulk-substance quality control. Thermodynamic data on protein aggregation and solubilization have been applied to formulation design of biologics. Consequently, their studies accelerated the growth of both structural biology and virology.

Northrop performed fractionated (NH4)2SO4 salting-out at 0 °C and μ ≈ 0.15, precipitating pepsin crystals isoelectrically at pH 1.5 and calculating a molecular mass of 3.4×10^4 Da from scattering coefficients. For trypsin crystals he analyzed activity changes before and after disulfide reduction, showing that the active site resides within the protein interior. Stanley’s TMV preparations achieved an A260/A280 of 1.2 and retained infectivity in crystal form, supporting the contemporary view that viruses are primarily proteinaceous. Their crystallographic data served as templates for Franklin’s fiber diffraction studies, allowing helical parameters (u = 49 Å, 16.3 subunits/turn) to be derived from toroidal patterns. These works remain foundational references for Kaufman’s thermodynamics of protein aggregation and McPherson’s kinetic models of crystal growth.