The ceaseless quest for scientific explanations for the origins of life only started with Louis Pasteur, the French chemist who, a century and a half ago, proved that living beings are born, necessarily, from their own species – in the 19th century, it was believed that fungi, insects and scorpions could emerge spontaneously from trash or from rotting animals. The doubts on the origin of life that persist today, oddly enough, point back to a chemical property discovered by Pasteur: there are molecules made up of the same elements and chemical links, but they appear in two distinct types – one to the left, the other to the right -, as if faced by their own image, reflected in the looking glass.
A phenomenon that involves these mirror-image forms and still intrigues scientists is the formation of proteins, molecules formed by smaller blocks, the amino acids, and essential for any living being. They are part of the structure of the chromosomes, they control the expression of genes, they make the division of cells viable, they regulate the production of energy, they act in the differentiation of the cells that are going to form tissues, they take part in the development of the embryo, and, in short, they determine how the organisms are going to be formed, grow, and die.
Well, only the left-handed amino acids take part in the formation of proteins, the ones chemically called levorotatory, or simply L. The right-handed molecules, known as dextrorotatory, or D., are simply eliminated from the game, although the two forms appear together and in equal proportions in nature. But how is it that the amino acids are separated, before forming the proteins, and why is it that only the left-handed ones triumph? Chemist Marcos Eberlin, from the State University of Campinas (Unicamp), has a few answers for the first part of the question.
Using mass spectrometry, a technique for chemical analysis that makes it possible to visualize with precision the molecular universe, Eberlin’s team and the team of an American called Robert Cooks, from Purdue University, in the United States, detected a process that had never been observed before: the attraction of left-handed amino acids (L) and right -handed(D) by their peers. This kind of organization, which makes it possible to understand the separation between these two molecules at the moment prior to the synthesis of proteins, is mainly due to geometry: the fits are so selective that they prevent the L amino acids from mixing with the Ds. Both the left-handed and right-handed amino acids form cylindrical structures, differentiated only by the fact of being mirror images, with eight amino acids of the same type.
They are the so-called octamers, like those in the illustration on this page, which shows serine, one of the 20 essential animo acids that, in different combinations, form thousands of proteins in plants and animals. Octamers are connected up to each other by means of the so-called hydrogen bonds, such as those that keep molecules of water together and from larger and larger structures. “It was, literally, a find, a first step towards understanding why only the L molecules take part in the formation of proteins”, comments Eberlin, who coordinates the Thomson Mass Spectrometry Laboratory at the Institute of Chemistry.
The information provided by the spectrometer and the computer simulations carried out by Fábio Gozzo, a chemist from the Thomson Laboratory team, makes it possible to see the agglomerates of serine, as if the molecules were playing ring-around-a-rosy, offering only the left hand, in this case the Ls, to the sockets made in the sides of the cylindrical structure. If one of the members leaves the group and the replacement can only offer the right hand, the chain is broken. “The process of chemical selection by the amino acids is a consequence of this perfect three-dimensional arrangement”, Eberlin points out. “All the molecules point in the same direction. If the dynamics are altered, the circle is not perfectly joined together. That is why the different ones are excluded”.
Another surprise was to observe that the cylinder of serine L, put in contact with the L and D forms of another amino acid, would capture only the L molecules. Described in an article published in May in the international edition of the German magazine Angewandte Chemie, this process – also unheard -of – won the name of chirality transmission. Chirality (from the Greek for ‘hand’, kheir) is the phenomenon described by Pasteur with tartaric acid: the molecules, when overlaid, form a pattern similar to the one that appears when we put one hand besides the other, with the palms and thumbs pointing upwards. “We are also observing an unprecedented mechanism for propagating the chirality of serine to other natural chiral amino acids, such as cysteine, asparagine, leucine, aspartate and methionine”, Eberlin explains.
Pollutants and blood
The discoveries were only made feasible with the improvements in mass spectrometry, a versatile technique applied, for example, in anti-doping, in quality control of fuels, and even in the sequencing of the set of proteins of an organism (proteome). With its use, Eberlin’s team is not only revealing the organization of the molecules essential for life. They are also expanding the use of the technique, with one innovation after the other: one of the most recent is a method for analyzing pollutants, derived from conventional mass spectrometry, but with an innovation: it uses a fiber coated with silicone that absorbs pollutants from the air, from steam and from liquids, after only one minute exposure. The fiber facilitates the capture, transport and assessment of the substances, afterwards transferred to the spectrometer, where they are examined. The description of this technique, called Fiber Introduction Mass Spectrometry (Fims), will be published shortly in Analytical Chemistry.
Fims is the latest offspring of other techniques for analyzing pollutants developed in the laboratory itself. The first of them is CryoTrap Membrane Introduction Mass Spectrometry (CT-Mims), which avails itself of a silicone membrane coupled to the spectrometer, to separate the pollutants and retain the water. This process increases sensitivity by up to one hundred times, and in a few minutes one can discover the concentration in water of chloroform, benzene and toluene, for example.
There are also innovations directly connected with human health. Last year, a partnership with Nelci Fenalti Höehr, from the Faculty of Medical Sciences (FCM) at Unicamp, resulted in the use of a derivative of a technique created in the laboratory itself, for determining the quantity of cysteine and of homocysteine in the blood. The two amino acids, which, in high concentrations, represent a risk factor for cardiovascular diseases, are captured from the blood plasma by the silicone membrane. Nelci sends the blood samples to the Institute of Chemistry, and the spectrometer gives the results in minutes. “Our target is to offer this service as a routine in the hospital complex at the School of Medical Sciences”, explains Nelci.
“Having overcome the limitations, we can today work with any kind of molecule, right from the small ones up to polymers, salts, sugars, peptides, proteins and gigantic molecules, even an intact virus”, says Eberlin. Without a doubt, quite an advance for a technique created at the beginning of last century by British physicist Joseph John Thomson (1856-1940), the same one who inspired the name for Unicamp’s laboratory. Indispensable in chemistry and physics laboratories since it arose, mass spectrometry makes it possible to isolate atomic and molecular structures, provided they are isolated, hence in a gaseous form, and ionized (with a positive or negative charge). This makes it possible to measure their properties, such as mass, chemical bonds, acidity, reactivity and structures.
Pilot in peril
To start with, the order of the procedures adopted during the analyses limited the applications: observation could only take place when the molecules are volatilized and are given a positive or negative electrical charge. This procedure is essential, because only molecules with positive or negative charges can be separated and directed – the neutral ones escape control. Until the beginning of the 90s, the molecule first had to vaporize inside the equipment, at a temperature of up to 300ºC, to be ionized only afterwards. The analyses were restricted to volatilizable and relatively small organic substances. Sugars or proteins were out of the question, they decompose when heated, before reaching the vapor stage.
To make headway, the order had to be reversed – first ionizing and then volatilizing. This was what American chemist John Fenn, now with the Virginia Commonwealth University, United States, did at the end of the 80s, when he created the Electrospray technique. This allowed the researchers from Unicamp and Purdue to discover the forms in which the amino acids are organized. First, they put the serine molecules into water.
With the solution, they made an electrolytic spray (a sort of aerosol made up of miniscule drops charged with a positive or negative charge). With the heating up and the evaporation of the water, the drops diminished in volume – and the serine octamers started to repel reach others so strongly that, at a certain moment,they were literally ejected from the drops to the gaseous stage – without decomposing, which would certainly have happened in the former technique for ionization. “It is a process that has a certain similarity with what happens when the pilot of an aircraft senses danger and ejects himself from the aircraft into space”, is Eberlin’s comparison.
Both the left-handed and the right-handed versions of serine took part in the experiment. As both of them show the same molecular mass, a carbon isotope of mass 13 was added to the L serine, as a way of differentiating the two forms. Put into water, in a random manner, the molecules surprisingly recognized their likes and gave rise to two sets of eight molecules, the octamers, one containing only D serines and others gathering together only the Ls. The researchers also observed the bond between twin octamers, which originate structures that have 16, then 32 and after that 64 amino acids – and so on successively. “For serine, the magic number is eight”, explains Eberlin.
The advances in mass spectrometry may also resolve the second part of the question: why do living organisms select only the set of left amino acids in the construction of proteins, leaving the right ones just to watch the game? In search of the answer, the team led by Arnaldo Naves de Brito, from the National Synchrotron Light Laboratory (LNLS), is working in collaboration with the Thomson Laboratory. Confident and enthusiastic, Brito says that the results of the study should be known shortly.
The difficulty is not only to reproduce the original terrestrial environment, when separation and selection would have happened for the first time. The bigger obstacle is the chemical and physical properties of the L and D molecules – they are almost identical and do not clearly show the paths of investigation to be followed. Even another scenario is considered for the primordial fits. “The selection of amino acids may have taken place on Earth or in another region of the universe, like meteorites or comets, to arrive here afterwards”, explains Ricardo de Carvalho Ferreira, a researcher from the Federal University of Pernambuco (UFPE), who back in the 50s was already working on the study of the chiral systems. In spite of the pitfalls, the task can be gratifying. “We are trying to understand the chemical architecture of living beings”, says Eberlin.
Modern Techniques in Mass Spectrometry and their Applications in Chemistry and Biochemistry (nº 00/11176-2); Modality Thematic project; Coordinator Marcos Nogueira Eberlin – Institute of Chemistry at Unicamp; Investment R$ 1,211,117.27