Electrophoresis and X-ray Diffraction

The development of new chemical and physical tools during the first half of the twentieth century made it possible for biochemists to probe with increasing finesse the very large protein molecules that seemed to be the very essence of life. In fact, what amounted to a new field of science, one that combined physics, chemistry, and biology, took for its realm of study the analysis of the fine structure and detailed functioning of the giant molecules of life. This new field, molecular biology, has become particularly important (and, indeed, quite spectacular in its achievements) since World War II, and has tended to overshadow the remainder of biology.

In 1923, the Swedish chemist, Theodor Svedberg (1884- ), introduced a powerful method for determining the size of protein molecules. This was an "ultracentrifuge," a spinning vessel that produced centrifugal force hundreds of thousands of times as intense as that of ordinary gravity. The thermal agitation of molecules of water at ordinary temperature suffice to keep the giant protein molecules in even suspension against the pull of ordinary gravity but not against such a centrifugal force. In the whirling ultracentrifuge, protein molecules begin to settle out, or "to sediment." From the sedimentation rate, the molecular weight of protein molecules can be determined. A protein of average size, such as hemoglobin, the red coloring matter of blood, has a molecular weight of 67,000. It is 3700 times as large as a water molecule, which has a molecular weight of only 18. Other protein molecules are larger still, with molecular weights in the hundreds of thousands.

The size and complexity of the protein molecule means that there is ample room on the molecular surface for atom groupings capable of carrying electric charges. Each protein has its own pattern of positive and negative charges on its molecular surface—a pattern different from that of any other protein and one capable of changing in fixed manner with changes in the acidity of the surrounding medium.

If a protein solution is placed in an electric field, the individual protein molecules travel toward either the positive or negative electrode at a fixed speed dictated by the pattern of the electric charge, the size and shape of the molecule and so on. No two varieties of protein would travel at precisely the same speed under all conditions.

In 1937, the Swedish chemist, Arne Wilhelm Kaurin Tiselius (1902- ), a student of Svedberg's, devised an apparatus to take advantage of this. This consisted of a special tube arranged like a rectangular U, within which a protein mixture could move in response to an electric field. (Such motion is called "electrophoresis.") Since the various components of the mixture moved each at its own rate, there was a gradual separation. The rectangular-U tube consisted of portions that fitted together at specially ground joints, and these portions could be slid apart. Matters could be arranged so that one of the mixture of proteins would be present in one component of the chambers and could thus be separated from the rest.

Furthermore, by the use of appropriate cylindrical lenses, it became possible to follow the process of separation by taking advantage of changes in the way light was refracted on passing through the suspended mixture as the protein concentration changed. The changes in refraction could be photographed as a wavelike pattern which could then be used to calculate the quantity of each type of protein present in the mixture.

The proteins in blood plasma, in particular, were subjected to electrophoresis and studied. They were separated into numerous fractions, including an albumin, and three groups of globulins, distinguished by Greek letters as alpha, beta, and gamma. The gamma-globulin fraction was found to contain the antibodies. During the 1940s, methods were devised to produce the different protein fractions in quantity.

Ultracentrifugation and electrophoresis depended upon the properties of the protein molecule as a whole. The us of X rays enabled the biochemist to probe within the molecule. An X-ray beam is scattered in passing through matter, and where the constituent particles of matter are arranged in regular ranks and files (as atoms are arranged within crystals) the scattering is regular, too. An X-ray beam impinging upon a photographic film, after being scattered by a crystal, appears as a symmetrical pattern of dots from which the arrangement and distance of separation of the atoms within a crystal may be deduced.

It often happens that large molecules are built up of smaller units which are arranged regularly within the molecules. This is true, for instance, of proteins, which are built up of amino acids. The regular arrangement of amino acids within a protein molecule is reflected in the manner in which an X-ray beam is scattered. The resulting scattering is less clear cut than that produced by a crystal, but it is capable of analysis. In the early 1930s, the general spacing of amino acid units was deduced.

This was sharpened in 1951, when the American chemist, Linus Pauling (1901- ), worked out the amino acid arrangement and showed that the chain of these units was arranged in the form of a helix. (A helix is the shape of what is usually called a spiral staircase.)

As men probed more and more deeply into the details of protein structure, it became necessary to deal with more and more complicated X-ray data, and the necessary mathematical computations grew long-winded and intractable, reaching a point where their detailed solution by the unaided human mind was impractical. Fortunately, by the 1950s, electronic computers had been developed which could perform routine computation of immense length in very little time.

The computer was first put to use in this manner in a problem involving not a protein, but a vitamin. In 1924, two American physicians, George Richards Minot (1885-1950) and William Parry Murphy (1892- ), had discovered that the regular feeding of liver kept patients from dying of a disease called "pernicious anemia." The presence of a vitamin was suspected. It was named vitamin B12 and in 1948 it was finally isolated. It proved to have a very complicated molecule built up of 183 atoms of six different elements. With the new physical techniques and the aid of a computer, the detailed structure of the vitamin was worked out in 1956. Because it was found to contain a cyanide group, a cobalt atom, and an amine group (among numerous other structures), it was renamed "cyanocobalamine."

It was inevitable that computers be applied to the diffraction patterns set up by proteins. Using X-ray diffraction and computers, the Austrian-British biochemist, Max Ferdinand Perutz (1914- ) and the English biochemist, John Cowdery Kendrew (1917- ), were able to announce, in i960, a complete three-dimensional picture of the molecule of myoglobin (a muscle protein something like hemoglobin but one quarter the size) with every amino acid in place.