Nonenzymatic Modifications to Keratins by Ethanol

It has been reported that in the aldehydic and/or glucose modification of proteins, only some lysine residues of the protein molecule are modified (Harding et al., 1989; Monnier et al., 1992; Vlassara et al., 1994). The reasons for these dissimilar results appear to reflect the structural differences in the amino acid residues surrounding the lysine residues. The first step in the glucose-mediated reaction is the formation of an aldimine followed by a rearrangement of the reaction product to an Amadori product and a set of further reactions, as demonstrated in Figure 8.3. As a consequence of these reactions, there is a decrease in the number of free amino groups in the protein and an increase in the effective negative charge of the modified protein. Thus, provided a sufficient number of modifications have occurred along the polypeptide chain, these modified proteins will migrate more swiftly to the anode. However, modification of the positively charged lysine residues will change the hydrophobic properties of the protein as well (Reiser et al., 1992).

An example of the effects of aldehydic changes was obtained by monitoring the effect of ethanol ingestion on the keratin taken from rat hair (Jelinkovâ et al., 1995). In this study CE profiles of hair keratins taken from rats fed 10% ethanol and from rats fed water were compared by single-dimension

OH I

R-N H-CH2-C-R' Amadori product

R-NH-CH2-COOH

Reactive intermediates

HOCH

HOCH

Pynaline

Furans Pyrrolidines Pyrroles Imidazoles

Pynaline

Advanced Maillaid Products

Figure 8_3 Scheme of the Miallard reaction.

electrophoresis (Fig. 8.4). The peaks can be placed in two groups: the peaks in the first group, with a retention time of about 5 minutes (two in the controls and three in the ethanol-treated animals) correspond to the fast-moving (narrow) zone shown in panel A\. The peaks in the second group, moving with the retention time of 7.5 to 10 minutes, and additional peaks moving with retentions of 15 to 18 minutes, correspond to the other (broad) zone seen panel A\.

In contrast to the results with single-dimension electrophoresis are the findings reported from two-dimensional gel separations (lower panels of Fig. 8.4). Two groups of spots are observed: a group of keratin proteins moving fast in the cathodic direction and a group distributed along a digonal. The literature suggests the two fast-moving peaks in the controls and the three in the ethanol-fed rats are low-sulfur keratins, while those in the second group, the diagonal smear, are high-sulfur keratin proteins.

This conclusion can be tested by passing the keratin proteins through a cation-exchange cartridge that retains only low-sulfur proteins. The retained fraction (released by washing the cartridge with 0.1 M triethylamine followed by overnight dialysis of the filtrate) corresponds to the first two (or three in alcohol-treated animals) fractions appearing during the capillary electrophoresis runs (see Figure 8.5AC). The unretained proteins correspond to the remaining, more anodically moving peaks. Indeed, amino acid analysis of the unretained fraction indicated a higher proportion of sulfur-containing amino acids in the unretained fraction, justifying the conclusion that the proteins in group 1 are low-sulfur and those in group 2 are high-sulfur proteins.

As mentioned above, the profiles in Figure 8.4 also indicate the presence of an additional low-sulfur component (arrow) in the alcohol-fed animals. This additional component has an apparent relative molecular mass of 70,000.

Further investigation of the high-sulfur proteins by two-dimensional electrophoresis under alkaline conditions was not possible because of smearing in the high-sulfur protein zone. Therefore two-dimensional electrophoresis separations were run at pH (Fig. 8.6). These separations showed two spots in the 25,000-35,000 relative molecular mass region. In CZE separations (Fig. 8.6A,B) run at pH 3.5, two distinct peaks appeared between 10 and 20 minutes running time in samples obtained from the alcohol-treated animals (indicated by arrows). These peaks were absent in control rats. When the fraction retained in the ion-exchange cartridge was run under similar conditions, the results seen in Figure 8.6C were obtained. By comparing the two sets of results, it is possible to identify the peaks moving within 12 minutes and less as low sulfur. Also, peaks moving more rapidly to the anode represent the high-sulfur fraction. This type of analysis leads to the conclusion that of three additional peaks of the alcohol-treated animals, two belong to the high-sulfur proteins, while the third fraction may be classified as low in sulfur.

Further characterization of the remaining fraction in the low-sulfur keratin group in the ethanol-treated animals included analysis of the trypsin-released peptides by plasma desorption mass spectrometry. These results indicated the

Absorbanc« (220 nm)

Absorbanc« (220 nm)

high sulphur low sulphur high sulphur high sulphur presence of a peptide differing in its molecular mass by three acetyl residues from another peptide found in controls. This finding suggests that three lysine residues were modified through the reaction with acetaldehyde.

The foregoing analysis makes clear that capillary electrophoresis can reveal differences in keratin proteins obtained from animals treated with ethanol compared to controls. However, it is also apparent that because of the complexity of the protein matrix, other methods must be used as well to identify the modified fraction.

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