New mass record

Professor Albert J.R. Heck of the Department of Biomolecular Mass Spectrometry of Utrecht University and Dr Willem J.H. van Berkel of the Laboratory of Biochemistry of Wageningen University have reported in Protein Science¹ on ground-breaking results in analysing very high molecular mass molecules. Together, they have managed to determine, using electrospray mass spectrometry, the molecular mass of the enzyme vanillyl-alcohol oxidase with great accuracy. This enzyme is of interest for the food industry because it is capable of producing the flavour compound vanillin (the main component of the vanilla aroma). The enzyme is also important scientifically, because it is considered as the prototype of a widespread family of structurally-related oxidoreductases.

By using nanoflow electrospray ionisation, the researchers from Utrecht and Wageningen were able to establish that vanillyl-alcohol oxidase is mainly composed of octamers with a molecular mass around 510,000 Dalton (see Figure), but that it also exists as 16-mer assemblies of 1.02 million Dalton and even as 24-mer assemblies of 1.53 million Dalton. This shows that this kind of bioactive protein complexes can even be larger/heavier than certain viruses. Apart from the remarkable fact that this type of large protein assemblies can be transferred and studied in the gas phase, it is remarkable that the uncertainty of the mass determinations is below 0.15%, many times more accurate than traditional methods such as gel filtration, ultracentrifugation and light scattering.

The mass spectrum and structure of vanillyl-alcohol oxidase, demonstrating detection of extremely high mass compounds.

These results have important implications for future research towards the structure and function of large protein complexes because up to now, the accurate determination of molecular masses was limited to less than hundred thousand Dalton.

Dr Willem J.H. van Berkel, willem.vanberkel@fad.bc.wau.nl, http://gcg.tran.wau.nl/local/biochem/ or Professor Albert J. R. Heck, a.j.r.heck@chem.uu.nl, www.chem.uu.nl/amsmass/www/amsmass.html.

Reference

1. W.J.H. van Berkel, R.H.H. van den Heuvel, C. Versluis and A.J.R. Heck, "Detection of intact megadalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry" Protein Science 9, 435 (2000).

Large organic molecules in space

Chemical synthesis of complex organic molecules can occur rapidly in stellar environments, according to results obtained with the European Space Agency's infrared space observatory, ISO.

Sun Kwok and Kevin Wolk, from the University of Calgary, Canada, and Bruce Hrivnak, at Valparaiso University, Indiana, USA, have studied the chemical composition of the circumstellar envelopes of old stars. They chose three types of old stars that are representative of three different stages of evolution, separated by just a few thousand years:

• very evolved red giants-the first evolutionary step
• proto-planetary nebula-the second stage;
• finally, planetary nebula.

The Water Lily Nebula in the constellation of Ara, one of the proto-planetary nebulae where complex organic molecules have been found. Photo: ESA.

By comparing their infrared spectra, the researchers could trace the processes of chemical synthesis leading to different compounds in each stage of the stellar evolution.

They found that several thousand years are enough for small organic molecules to evolve into large, complex organic molecules. For instance acetylene, which is detected in the envelope of red giants, serves as a building block for molecules such as benzene and more complicated aromatic hydrocarbons present in the planetary nebula.

"Although we do not understand how chemical reactions can occur so efficiently in such a low density environment, there is no doubt that complex molecules exist, and the stars are able to make them with no difficulty", says Kwok.

According to Sun Kwok, the finding of complex organic molecules in stellar envelopes might provide an easier explanation for the beginning of life on Earth, since it is quite possible that some of these molecules will end up on planets. Kwok also suggests that even amino acids could be synthesised in the stellar environments, although to look for them astronomers will have to wait for future infrared space telescopes such as the European Space Agency's Far Infrared and Submillimetre Telescope (FIRST), to be launched in 2007.

One of the infrared spectra from ISO of proto-planetary nebula, IRAS 22272+5435, showing several examples of aromatic and aliphatic features. N.B. This is not the same object as in the image above. Image: ESA.

Further work on ISO by a group of Spanish astronomers has lead to the detection, for the first time outside the Solar System, of two molecules that could be the precursors for the formation of complex organic compounds. The newly-found molecules, detected in two very old stars, are diacetylene and triacetylene (C₄H₂ and C₆H₂). This adds information about the intermediate chemical steps that lead from one of the simplest organic molecules, such as acetylene, to the complex PAHs discovered by Sun Kwok, Kevin Wolk and Bruce Hrivnak.

"ISO has provided an important database for the study of these large and complex organic molecules", says José Cernicharo, from the Centro Superior de Investigaciones Científicas (CSIC) in Madrid. "This will allow us to investigate their role in the chemistry of interstellar space, and to answer important questions that remain open. For instance, how are these large species formed?"

Using ISO, Cernicharo and co-workers observed two stars in the process of dying, CRL618 and CRL2688, which have been blasting out huge amounts of material over the last thousand years and thus have become the central stars illuminating a large shell of gas and dust-a structure called "a proto-planetary nebula". The astronomers studied the chemical composition of the gas around the stars and realised that many new molecules had been synthesised. Many of these molecules are unknown, but the researchers were able to identify at least two of them: C₄H₂ and C₆H₂, di- and tri- acetylene.

"The large number of unknown molecular bands revealed by ISO left us astonished. Among them we quickly identified two new molecules, di- and tri-acetylene, which are present in the planets of the Solar System but had not been found before in the interstellar space. The unknown molecular species and the di- and tri-acetylene might very well be the 'small bricks' that will combine to make the complex molecules like PAHs", Cernicharo explains.

In the proto-planetary nebula CRL618 Cernicharo and Fabrice Herpin (CSIC) have found also water (H₂O) and OH, an unexpected result because CRL618 is a carbon-rich object and those are oxygen-bearing molecules. These are examples of how powerful the stars are when it comes to the production of new molecules... molecules that may end up in planets like the Earth.

As Cernicharo explains, "when an old star is evolving towards the proto-planetary phase, it produces violent phenomena such as high velocity winds and high flux of high energy photons; these phenomena modify completely the chemistry of the gas around the star, and allow the formation of new molecules. With time, they will escape from the gravity of the central star and will reach the interstellar medium, where they will joint the molecular clouds out of which new stars will form. When a new star with its planetary system is formed, highly complex molecular species, many of them containing a large number of carbon atoms, are already present to form part of comets and planets".

Herschel exhibition

This year is the 200th anniversary of the discovery of near infrared radition by William Herschel: see the article starting on page 10 for more information. Herschel is probably more widely known for his work as an astronomer and his discovery of Uranus. Indeed it was while trying to cut down on the heat coming from his telescope that he started the experiment that led to the discovery of NIR!

One of the pictures in the Herschel Exhibition. The constellation of Orion seen in the infrared; data collected by IRAS, the Infrared Astronomical Satellite. This picture is a composite of IRAS wavelength band data centred at 12 mm, 60 mm and 100 mm. The warmest features, e.g. the stars, are brightest at 12 mm. The interstellar dust is cooler and shines brighter at 60 mm and 100 mm. Photo courtesy of Infrared Processing and Analysis Center, Caltech/JPL.

To celebrate the anniversary, an exhibition of infrared images of the Universe has been assembled by Dr Helen Walker of Rutherford Appleton Laboratory, Didcot, UK. This exhibition will be touring the UK during this year. Contact Dr Walker for dates and locations h.j.walker@rl.ac.uk.