Applications To Biological Kinetics

As discussed below, there are only a few examples of the use of synchrotron XRD techniques to study food process kinetics. Therefore, the potential of the method will be illustrated first using examples drawn mainly from biological studies, where there is a substantial history of this kind of work. Such studies include: (1) protein catalysis, initiated using stopped-flow or 'caged' metabolite techniques, including reactions that can be induced to take place within protein crystals; (2) structures of proteins in solution, such as soya globulin; (3) structural kinetics, e.g. the assembly of microtubules in biological cells, where the process is initiated by a change of temperature; or the assembly of a plant virus; (4) lipid crystallization events which are initiated by a rapid change of temperature or pressure ('temperature-jump' or 'pressure-jump' experiments); and (5) crystallization that is induced by shear, as in the study of industrial polymers.

3.1. Protein catalysis mechanisms

Mechanisms of catalysis within a protein crystal can be characterized if the process is synchronized over all the protein molecules. In order to synchronize events, an inactive, photolabile precursor is allowed to diffuse into the crystal over a period of time and then subjected to a flash of laser light, in order to release the active metabolite all over the crystal simultaneously (Bartunik & Bartunik, 1992). This approach relies on the large amount of water in a crystal, which can occupy well over half the total volume. Other approaches for triggering structural catalysis in a crystal include rapid temperature change (T-jump); release of protons ("caged" protons); and optical "pumping". Using the same methods, catalysis also can be studied in solution. Studies of events that require milliseconds are being carried out. The third-generation synchrotron radiation sources currently being built will facilitate the study of events that occur in microseconds (non-cyclic events) or even nano-seconds (cyclic events).

Another example is one of optical pumping. In this case, cyclic structural changes occurring in a membrane protein have been investigated by XRD (Koch et al., 1991). The protein, called bacteriorhodopsin, uses light energy to pump protons across a cell membrane. The absorbing chromophore is a protein to which a retinal molecule is bound covalently. Two different protein structural changes, occurring in seconds and milliseconds after light is absorbed, have been characterized and compared to optical spectroscopy observations.

3.2. Protein structure/dynamics in solution

The 11S soya globulin has been characterized in a 5% solution, using synchrotron radiation (Miles et al., 1984). The measurements are more rapid than for conventional sources by 100- to 1000-fold. The shorter exposure times forestalled well known structural changes ~ aggregation and/or denaturation — that occur during the longer time required for conventional XRD exposures.

Muscle has been studied extensively by XRD, and synchrotron exposures have greatly improved the time resolution of the structural kinetics of contraction. In ancillary studies, a muscle protein, troponin C, has been studied in solution in order to understand the role of Ca in contraction (Ueki, 1991).

3.3. Assembly of organelles

As an example of characterizing structural kinetics in solution, the growth of virus rods from protein monomers has been characterized using synchrotron radiation (Ueki, 1991).

3.4. Structural kinetics in phospholipid membranes

Rapid crystallization events in phospholipid membranes have been characterized using synchrotron radiation. The events have been initiated by gradual heating and cooling, at rates of the order of 5°C/minute (Quinn, 1992); or by a very rapid drop in pressure, which can have a similar effect to very rapid heating (Caffrey, 1991).

3.5. Crystallization under shear

A study of direct relevance to many food processes is crystallization under shear. Under conditions where a quiescent sample of plastic takes an hour to begin to crystallize, the process begins in seconds under even a low shear (Moitzi and Skalicky, 1993).

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