Glutaraldehyde penetration

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

A high diffusion coefficient increases the rate of diffusion, all else being the same. The diffusion coefficient is determined in part by molecular size and shape. Small molecules tend to have high diffusion coefficients, which is one reason why formaldehyde penetrates faster than glutaraldehyde. In addition, interactions between the chemical and its environment will influence the diffusion coefficient. Thus, if the chemical hydrogen bonds to the water around it, the diffusion coefficient will be lower and the rate of diffusion will be reduced. 

Penetration of the biomembrane is undoubtedly essential for most membrane activity. Araki and Rifkind (13) obtained esr spectra of stearic acid spin labelled erythrocyte membranes in the presence of diverse compounds including Triton XlOO, chlorpromazine and glutaraldehyde. The two surfactants chlorpromazine and Triton XlOO both increase the rate of haemolysis and are shown to increase membrane fluidity. Glutaraldehyde as expected decreases fluidity and decreases the rate of haemolysis. 

Formaldehyde, a monoaldehyde, is preferred over glutaraldehyde (a dialdehyde) because the latter introduces mostly irreversible protein crosslinks, masking the epitopes. Formaldehyde penetrates rapidly into the tissue but crosslinks proteins slowly. Most of these crosslinks are reversible, and therefore masked epitopes can be easily unmasked by treatments such as heating. However, for better preservation of cell morphology, a mixture of formaldehyde (4%) and glutaraldehyde (0.01-0.1%) can be tried. Bouin s fixative with its acidic pH should not be used. 

As explained in this chapter, glutaraldehyde introduces mostly irreversible protein crosslinks that may alter the conformation of the antigen (epitope) molecule. Such extensive crosslinkages become a barrier to the antibody penetration, and thus its accessibility to the epitope is hindered. This impediment becomes a serious problem when monoclonal antibodies specific for only one epitope type are used and/or when epitopes are not located at the surface of the antigen molecule. 

Glutaraldehyde crosslinks proteins and may alter protein configuration and mask epitopes. Because of its cross-linking properties, penetration of reagents may be impaired. For immunocytochemistry, it is usually used in low concentration in combination with formaldehyde. 

Fix dissected adult tissues or embryos in fresh 4% paraformaldehyde/0.2% glutaraldehyde in 0.1 M phosphate buffer. Determine the time required by the size of the tissue to be fixed based on penetration rates of 1 mm/h at room temperature. (Perfusion can also be carried out if desired, see section 1.3.2.2),... 

For an excellent review of tissue and section preparation methods in plants, see ref. 31. Fixation conditions will vary with the tissue, the cell-type and its permeability to the fixative. Glutaraldehyde does not easily penetrate leaf cuticle, but is immediately available to stem cross-sections. Fixation with a 2.5% glutaraldehyde in 0.1 M Na2HP04 pH 7.0 for 2 to 3 minutes on ice seems to leave a reasonable amount of GUS activity, when followed by extensive washing. Fixation should be tested empirically in any new system. Formaldehyde seems to be a more gentle fixative than glutaraldehyde, and can be used for longer times. We have successfully used 0.3% formaldehyde in 0.3 M mannitol, 10 mM MES, pH 5.6 for 30 to 45 minutes at room temperature for protoplasts. This should be followed by several washes, usually in phosphate buffer or osmoticum. 

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