The Effect of Reactive Aldehydes on Free Primary Amino Groups in Collagen

In unmodified type I collagen, the number of free primary amino groups, as determined by the assay, was linearly related to the collagen concentration when the protein was present at a concentration between 0 and 50 pg/ml (Figure 2.1). Appendix 1 shows the raw data.

The % free primary amino groups in collagen decreased as the malondialdehyde concentration, to which it was exposed, was increased (Figure 2.2). A 50 % decrease in the number of free amino groups was obtained at a malondialdehyde concentration of approximately 100 mM. Two way analysis of variance showed that the effect of malondialdehyde concentration on % free primary amino groups was significant (p = 0.0001).

A similar effect on the % free primary amino groups was achieved when 2,4-hexadienal was used as the reactive aldehyde to modify collagen instead of malondialdehyde (Figure 2.2). An increase in the concentration of 2,4-hexadienal caused a decrease in free primary amino groups. The hexadienal concentration had to be approximately 50 mM before a 50 % decrease in amino groups could be seen. The concentration of hexadienal had a significant effect on the loss of collagen amino groups (p = 0.001), as determined by two way analysis of variance.

The percentage of free primary amino groups in collagen decreased as the glutaraldehyde concentration increased. This effect was shown to be significant by two way analysis of variance (p = 0.0001). With glutaraldehyde, a 50 % decrease in the number of free collagen amino groups was obtained at a concentration of approximately 40 mM (Figure 2.2).

Figure 2.1

Relationship between Collagen Concentration and Free Primary Amino Groups as determined by the Fluorescamine Assay

Fluorescamine solution was added to borate buffer containing the collagen sample. The fluorescence, at = 390 nm and 465 nm, was read after two minutes.

Figure 2.2

Effect of Reactive Aldehydes on the Number of Free Primary Amino Groups in Collagen

Collagen was modified with malondialdehyde (MDA) ( * ) , hexadienal (HDA) (O ) or glutaraldehyde (OTA) (□ ). Fluorescamine solution was added to borate buffer, containing one of the three modified collagen samples. The fluorescence, at = 390 nm and = 465 nm, was read after 2 minutes.

f

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I

I

I

% Primary amino groups

J

è

8

8

8

1

fO

% Primary amino groups

8

8

8

8

8

8

8

8

At a concentration of 50 mM, all three reactive aldehydes caused a significant decrease in the number of free primary collagen amino groups (p < 0.01). However, glutaraldehyde caused the greatest loss in collagen amino groups and malondialdehyde the least.

The raw data can be seen in Appendices 2, 3 and 4.

2.3.2 Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was performed on native and aldehyde-treated collagen samples to characterize and compare any structural differences between the samples.

Native collagen a and P chains ran normally. The P chains were dimers composed of two a chains. There appeared to be approximately twice as much p i,l(I) as P 1,2(1). The collagen a2 chain had a higher electrophoretic mobility than the a l chain. A number of bands also appeared at the top of the gel for y and Ô chains, corresponding to trimers of three a chains and even higher relative molecular mass species (Fig. 2.3, Lane 1).

The reaction of malondialdehyde with collagen resulted in a very large increase of high molecular mass chains (the y and Ô bands). These ran as one very broad band of lower electrophoretic mobility than the separate and distinct sharp bands for native collagen. The p chains also ran as a broad band instead of the sharp p chain bands of native collagen. The p chains of malondialdehyde- treated collagen migrated approximately the same distance through the gel as the p chains of native, untreated collagen. The modification of collagen with malondialdehyde significantly decreased the number of low molecular weight a chains (Fig. 2.3, Lane 2).

Collagen which had been modified with hexadienal displayed an increase in higher molecular weight chains, compared to those for native collagen. In the same way as malondialdehyde-treated collagen, these chains ran as one band. However, the band was not as broad and had penetrated slightly further into the gel than for collagen pretreated with malondialdehyde. The p chains of hexadienal-treated collagen ran to approximately the same distance on the gel as both untreated collagen and malondialdehyde-treated collagen. They ran as two sharp bands, but the P chain of higher electrophoretic mobility was less intense than the comparable band for native collagen. The

49

a chains migrated to the same position as those for native collagen, but were of a greatly reduced intensity (Fig. 2.3, Lane 3).

The a, P and higher molecular weight chains of collagen which had been modified with glutaraldehyde had the same electrophoretic mobility as the comparable chains in native collagen. However, all of these bands were much less intense and the a chains had almost completely disappeared. A significant amount of the glutaraldehyde-treated collagen had not managed to penetrate far into the stacking gel and was unable to enter the separating gel (Fig. 2.3, Lane 4).

The increase in higher molecular weight species, together with a simultaneous decrease in the lower molecular weight a chains, suggests that new cross-links are formed when collagen is reacted with either malondialdehyde or hexadienal. Collagen reacted with glutaraldehyde resulted in an extensive increase in relative molecular mass subunits, inferring that many new intermolecular cross­ links were formed and that glutaraldehyde was more efficient at cross-linking collagen than malondialdehyde or hexadienal.

Figure 2.3

SDS Gel of Native and Modified Collagens

2 % SDS, 8 M urea, 0.05 % bromophenol blue, 0.1 % D IT and 100 ml 0.0625 M Tris-HCl (pH 6.8) were mixed to form a sample buffer.. Solutions of collagen modified with either malondialdehyde (Lane 2), hexadienal (Lane 3) or glutaraldehyde (Lane 4) were added to sample buffer in a ratio of 1:2 and boiled. The samples were run on a 5 % separating gel and a 7 % stacking gel. A native collagen solution (Lane 1) and Biorad high molecular weight markers were also run. Protein bands were visualized by silver staining.

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2 0 5

116

5

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