Molecular Biology: Protein. Chromatography

The use of physical methods, such as X-ray diffraction, to work out the detailed structure of a large molecule, is immeasurably aided if chemists have already determined the chemical nature of the subunits of the molecule and have obtained a general notion of their arrangement. If this is done, the number of possibilities into which the esoteric diffraction data need be fitted, is cut down to a practical size.

In the case of proteins, chemical progress was slow at first. The men of the nineteenth century had only been able to show that the protein molecule was built up out of amino acids. As the twentieth century opened, the German chemist, Emil Hermann Fischer (1852-1919), demonstrated the manner in which amino acids were combined within the protein molecule. In 1907, he was even able to put together fifteen molecules of one amino acid and three of another to form a very simple eighteen-unit proteinlike substance.

But what was the exact structure of the far more complicated protein molecules occurring in nature? To begin with, what was the exact number of each type of amino acid present in a given protein molecule? The straightforward method of answering that question would have been to break up the protein molecule into a mixture of individual amino acids and then to determine the relative quantities of each component by the methods of chemical analysis.

This procedure was impractical, however, for the chemists of Emil Fischer's day. Some of the amino acids were sufficiently similar in structure to defeat ordinary chemical methods intended for use in differentiating among them.

The answer to the problem came through a technique, the ancestor of which first saw the light of day in 1906, thanks to the labors of a Russian botanist, Mikhail Semenovich Tsvett (1872-1919). He was working with plant pigments and found a complex mixture on his hands, one made up of compounds so similar as to be separable only with the greatest difficulty by ordinary chemical methods. It occurred to him, however, to let a solution of the mixture trickle down a tube of powdered alumina. The different substances in the pigment mixture held to the surface of the powder particles with different degrees of strength. As the mixture was washed downward with fresh solvent, they separated; those components of the mixture which held with less strength being washed down further; in the end, the mixture was separated into individual pigments each with its own shade of color. The fact of separation was "written in color" and Tsvett named the technique, from the Greek for that phrase, as "chromatography."

Tsvett's work roused little interest at the time, but in the 1920s Willstatter reintroduced it and made it popular. Chromatography came to have a wide and varied use in the separation of complex mixtures. In the form of a tube of powder, however, it could only with difficulty be applied to very small quantities of mixture. Something still more powerful was needed.

The necessary modification came in 1944 and revolutionized biochemical technique. In that year, the English biochemists. Archer John Porter Martin (1910- ) and Richard Laurence Millington Synge (1914- ), worked out a technique for carrying on chromatography on simple filter paper.

A drop of an amino acid mixture was allowed to dry near the bottom of a strip of filter paper and a particular solvent (into which the bottom edge of the strip could be dipped) was then allowed to creep up the strip by capillary action. As the creeping solvent passed the dried mixture, the individual amino acids contained therein crept up with the solvent, but each at its own characteristic rate. In the end, the amino acids were separated. Their position on the paper could be detected by some suitable physical or chemical method and matched against the position of individual amino acids treated separately in the same way on other pieces of paper. The quantity of amino acids in each spot could be determined without much difficulty.

This technique of "paper chromatography" proved an instant success. Simply and inexpensively, without elaborate equipment, it neatly separated tiny amounts of complex mixtures. The technique was quickly applied to virtually every branch of biochemistry—to Calvin's work on mixtures in photosynthesizing plant cells, for instance—until research without the technique has become virtually unthinkable.

In particular, paper chromatography made it possible to determine the exact number of the different amino acids present in a particular protein. Protein after protein came to be characterized by the number of each of its constituent amino acids, as an ordinary compound might be identified by the number of atoms of each of its constituent elements.