A separate area is represented by the analysis of proteins, mainly proteins of connective tissue. The more significant results were the following.

1. Electrophoretic separation of proteins and their fragments in acidic buffers in the presence of high concentrations of surfactants. 
2. Separation of peptides and proteins (collagen) in polymer filled capillaries (polyacrylamide, cellulose or polymeric surfactant) or in highly acidic buffers. 
3. Electromigration separations of proteins based on the sorption equilibrium with the inner surface of the capillary (the proposed term for this mode is "open tubular capillary electrochromatography"). 
4. Identification of collagen fragments arising under the action of tissue collagenase (metalloproteinase) in lung hypoxia. 
5. Localisation of the fluorescent entities in the collagen molecule arising by nonenzymic glycation.

Methods which were developed for the separation of collagen fragments (capillary electrophoresis in acidic buffers) were successfully applied to quantitation of collagen type I, III and V in tissues.

All these analyses aimed at better understanding the physiological role of spontaneous nonenzymic (chemical) reactions between the free amino group (lysine, arginine, Nterminal amino acid) of slowly metabolised proteins and the oxo group of endogenous compounds (e.g. aldehydic sugars, lipid peroxidation products). These reactions have a significant effect in normal physiological (ageing) or pathophysiological (diabetes) situations, i.e. in changes of the biological functions of proteins. The next aim was the elucidation of the role of toxic substances capable of protein modification in the environment, in foods as well as revealing in the metabolically inert deposits of such compounds in tissues. A related aspect was a study of the kinetics of low molecular mass metabolites (including inorganic ions) and their binding to the extracellular matrix components. For this purpose a method capable of determining the binding of lead and zinc to collagen (or its fragments) was developed. It was demonstrated that two categories of binding sites exist in the collagen molecule, the number of which correlates rather well with the available aspartic and glutamic acid residues. It was also described that lead (after its administration in water) is accumulated in collagencontaining tissues (placenta and chorionic membranes).

High fat diet feeding of laboratory rats yielded a considerable increase in the concentration of reactive carbonylcontaining compounds in rat heart reperfusates (i.e. it resulted in an increase of reactive carbonyl compounds capable to react with proteins present in the vascular walls). The concentration of these metabolites can be brought back to control level when the high fat diet is switched back to standard pelleted diet.

Metabolites arising from posttranslational modifications (glycation) were found in specific (two) collagen fragments (revealed by capillary electrophoresis with offline coupled synchronous fluorescence spectroscopy). Collagen glycation and modification with lipidderived metabolites was studied in ageing and hypertriglyceridemic (HTG) animals. It was found that pentosidine (glycationspecific amino compound measured by HPLC) concentration significantly increases with age in collagen from rat skin and tail tendon but fluorescence (measured at four wavelengths typical of sugar or lipidderived adducts) significantly increase with age only in skin collagen. Significant differences between HTG and control animals were observed only in the pentosidine content for young animals; in old animals the values were identical. We noccluded that longlived proteins in different nocnective tissues are differently available for posttranslational modifications and that results obtained with tail tendons (as a typical connective tissue model) cannot be automatically applied to other soft connective tissues in the body.

Posttranslational modifications of proteins by alcohol intake were also investigated. This research was done in cooperation with the Institute of Forensic Medicine, University of Verona, Italy. There were monitored and discovered changes in structure (composition) of hair proteins (keratins) and serum transferrin which were induced by alcohol. These peptide modifications in transferrin have forensic (diagnostic) consequences that may be practically useful.