Szent-Gyorgyi had a simple hypothesis, that cancer could be reversed by restoring its ESR signal and healthy pink color, by means of decreasing the electron acceptor/donor quotient. Szent-Gyorgyi wrote:
White casein is a protein that is considered to be a poor electron acceptor until it reacts with methylglyoxal. Methylglyoxal changes pure white casein powder to a vivid brown color; brown casein is found to have about 100X larger ESR signal than untreated casein. The action of methylglyoxal was to desaturate the otherwise electron-filled ground states of the casein macromolecules, leading to a rearrangement of electrons in the outer orbital shells of the reagents, and a change in color and ESR signal.
"The malignant nature of cancer, due to its senseless proliferation, is intimately connected with the physical state that makes the cancer uncolored and makes it give a poor ESR signal. This makes it likely that the restoration of color and ESR signal to such cancer could also abolish malignancy."
Next, the investigators wanted to know which protein residues were altered in response to methylgloxal treatment. As outlined by the studies below, a special role was identified for lysine, the amino acid with a propylamine group. In the figure below, curve 1 is the absorption peak, with lambda max of 328 nm (yellow color), of methylgloxal-treated bovine serum albumin (BSA-MG).
Authors suggested that the peak at 328 nm (curve 1) could be a resonance stabilized n-pi* transition for a Schiff base linked to an aldehydic carbonyl group of methylglyoxal. The conversion of white to colored BSA indicates the formation of a Schiff base (-HC=N-) linkage between the amino group of lysine side chains and methylglyoxal. The involvement of a Schiff base in the color transition was demonstrated by chemically blocking the lysine side chains of BSA (curve 2, Figure 1 above), leading to a complex with no yellow color. This showed that the presence of lysine side-chains, probably nitrogen p orbitals, are essential for brown coloration in the proteins.
The appearance of the yellow color reaction was sensitive to pH. When the reaction was held at pH 4 (curve 3, Figure 1 above), there was no appearance of yellow color or an absorption peak at 328 nm, because at pH below 7, protonation of the lysine amine side groups is expected to prevent the color reaction and the formation of a protein-methylglyoxal complex.
In Figure 4 (below), the absorption peak at 330 nm was monitored in time, for a BSA-methylgloxal complex in a variable pH environment. A shift to pH 2 leads to a decrease of absorption, and while a shift to pH 7 increases the absorbance of the complex, further showing the relationship between visible color of proteins and their state of electronic desaturation.
Brown and white casein differed in electroconductive properties as well. Brown methylgloxal-treated BSA (BSA-MG) exhibited a larger D.C. conductivity than untreated BSA, as shown in the plot below.
"When proteins such as casein, collagen, bovine serum albumin, and lysozyme react with methylglyoxal they assume a stable brown color and exhibit a greatly enhanced electronic conductivity and ESR activity compared with the normal unreacted proteins." (J.A. McLaughlin, 1980)