The Interpretation of Electron Micrographs of Negatively Stained Native Collagen

Abstract

Deamination of native rat tail collagen followed by treatment with 1,2-cyclohexanedione removes the positive charge from the side chains of the lysine and hydroxylysine residues and blocks approximately 70% of the positively charged side chains of the arginine residues. When such specimens are examined in the electron microscope after negative staining with 2% potassium phosphotungstate at pH 6.8-7.4 no change from the appearance of normal collagen is detectable. These findings suggest that electrostatic attraction does not occur between the stain and the side chains of the lysine, hydroxylysine and arginine residues of the collagen and justify the view that, at least with this stain and under these conditions, the effect is that of pure negative staining and not that of a mixture of positive and negative staining. Furthermore, the experiments indicate that possible electrostatic attractions between the positively charged side chains of lysine, hydroxylysine and arginine and the negatively charged side chains of residues such as aspartic acid and glutamic acid are relatively unimportant in the maintenance of the light cross-striations of negatively stained collagen. However, the loss of these light cross-striations after treatment of collagen with urea implies that they are mainly associated with intermolecular hydrophobic and, possibly, hydrogen bonding.