STURE FORSÉN, LUND UNIVERSITY, SWEDEN
I came under the spell of calcium in a fairly roundabout way. During my graduate studies in the early 1960s, I was involved with nuclear magnetic resonance spectroscopy of various nuclei. 1H and 13C were, of course, the most popular since they usually gave rise to very narrow NMR signals and are present in almost all organic and biochemical molecules. These nuclei, however, represent only a minor fraction of the elements in the periodic table--most of which were more or less virgin territory for NMR. But many of these less popular nuclei are in fact quite abundant in biological systems--not the least in the form of ions, for example, Cl, Na+, K+, Mg2+, and Ca2+.
One reason these nuclei were relatively unpopular was that they possessed a property called "quadrupolar moment." Unless the quadrupolar nuclei are in an effectively symmetrical situation--like, for example, as "free" ions in dilute aqueous solutions--they give rise to very broad NMR signals. In covalent compounds, the linewidths can easily be 10,000 Hz or more. Furthermore, some of the isotopes suitable for NMR were in low abundance--for example, 25Mg (10.1%) and 43Ca (0.13%), and, in addition, their inherent NMR receptivity was generally poor.
Due to a mixture of scientific curiosity, stubbornness, and sheer instrumental necessity--lack of a spectrometer suitable for high-resolution 1H NMR studies of biological macromolecules--we used NMR of 35Cl, 79Br, 81Br, and 23Na+ to study how these ions interacted with different proteins, primarily hemoglobin and other heme proteins, metalloenzymes, and so forth. Much valuable information on the proteins could indeed be obtained through competition experiments with substrates, inhibitors, and other ions. This was largely due to a very useful dynamic amplification phenomenon. Even if a quadrupolar ion in an aqueous solution was binding to a metal ion or a charged amino acid on a protein molecule only for a very brief time, this resulted in an easily observed broadening of the NMR signal of the bulk ions due to chemical exchange that transferred the large linewidth of the bound ions to the bulk.
But we wanted to be able to move from the "easy" quadrupolar ions to the really tough ones: 39K+, 25Mg2+, and 43Ca2+ , the last being the real crown jewel. I was able to convince a major funding institution in Sweden to provide a grant to construct a superconducting Fourier transform NMR spectrometer dedicated to the study of quadrupolar ions in biological systems. This instrument was operational around 1980, and, as a result of the outstanding skill of electronics engineer Hans Lilja, was world-class. We were now ready for 43Ca2+ studies. In 1982, we were able to present in the Journal of the American Chemical Society our first--in fact the first--NMR study of 43Ca2+ at millimolar concentrations interacting with different Ca2+ binding proteins [J. Am. Chem. Soc., 104, 576 (1982)]. In subsequent studies of a variety of proteins, we showed how the rate of exchange, under equilibrium conditions, of the Ca2+ ions--as well as their binding constant--could be measured by varying the temperature and concentrations.
By 1980, it had become evident that in higher organisms intracellular Ca2+ was a universal "second messenger" involved in the activation of an enormous variety of cellular processes: muscular contraction, secretion, fertilization, apoptosis, and memory. As a result of events at the surface of a cell--a receptor meeting a signaling molecule, for example--the intracellular Ca2+ concentration was transiently increased from nanomolar to micromolar due to release of Ca2+ ions from intracellular storage devices. Several of these effects could be traced back to the binding of Ca2+ to an intracellular Ca2+ binding protein called calmodulin, discovered in the late 1970s. Calmodulin, like many other intracellular Ca2+ binding proteins that were subsequently discovered in rapid pace, possessed pairs of specialized binding sites called "EF hands." At micromolar Ca2+ concentrations, calmodulin would bind Ca2+ in a cooperative manner. As a result, calmodulin underwent conformation changes that enabled it to bind to and activate a large number of other protein molecules.
Today, many hundreds of Ca2+ binding proteins are known. They have a wide range of structures and functions--encompassing signaling, transporting, and buffering [Biochem. Mol. Biol., 36, 107 (2001)].
A recent Google search of "calcium" gave 3 million hits. Calcium rules!
Sture Forsén is professor emeritus at Lund University, Sweden, and managing director of a postgenomic consortium (SWEGENE). From 1976 to 1995, he was a member of the Nobel Committee for Chemistry of the Royal Swedish Academy of Sciences.
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