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Spectrophotometric and chromatographic studies on Spicer's high iron diamine reaction suggest that new red and blue oxidation products are formed, which become rapidly aggregated mainly to ferrous ions. Model experiments with pure substrates of known physical and chemical nature at pH 1.3-1.5 indicate that this reaction selectively demonstrates compounds carrying ester sulfate groups. Substrates with the highest charge density ( e.g., heparin and dextran sulfates) give dark purple or violet colors, whereas others with lower charge density give brownish reddish or purplish shades.

The thiocholine method of Koelle (1951) was used in attempts to demonstrate acetylcholinesterase activity in artificial enzyme-gelatin mixtures and in suspensions and smears of isolated platelet, ghosts, erythrocytes, and megakaryocytes of different species. When the last processing step with ammonium sulphide was omitted, the following observations were made: After a short time of incubation, small granular or rod-like deposits were found at the presumed sites of AChE activity. These deposits apparently grew into large crystals during prolonged incubation. Such crystals were demonstrated at the surface of platelet ghosts and erythrocytes of species bearing AChE activity, and further in megakaryocytes of some species presumably bearing this activity. The crystals were provisionally assumed to be a Cu thiocholine salt representing sites of AChE activity. If an enzyme inhibitor was added or a substrate used which is not split by AChE no crystals appeared.

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