Enzymic and immunochemical properties of lysozyme—VI Conformation, enzymic activity and immunochemistry of derivatives modified at arginine residues

Modification of the arginine residues in lysozyme with cyclohexanedione has been carried out under two different conditions. The first derivative (CHD-Lysoz I) was prepared by reaction in 0·1 N NaOH. However, it was found that control lysozyme preparations subjected to these reaction conditions lost all enzymic activity, although their antigenic reactivity was not affected. To avoid this complication, a second derivative (CHD-Lysoz II) was prepared by reaction in 0·1 M triethylamine. In fact, lysozyme pretreated with this solvent retained entirely its enzymic activity and its antigenic reactivity. In both CHD-Lysoz I and CHD-Lysoz II, 10 arginine residues were modified. The two derivatives possessed identical conformations, as revealed from their optical rotatory dispersion and circular dichroism behaviors and from the availability of the disulfide bonds to reduction and susceptibility to chymotryptic attack, but were greatly unfolded relative to lysozyme. The two derivatives had no enzymic activity and very little antigenic reactivity with antisera to lysozyme. Conversely, with antisera to CHD-Lysoz I, lysozyme reacted only partially (60 per cent), and these sera showed appreciable (28 per cent) cross-reaction with CHD-human serum albumin. Interpretation of the enzymic and immunochemical data was complicated by the fact that too many (10) arginine residues were modified and that appreciable conformational changes were present. Preparation of a derivative with a least number of modified arginine residues was therefore desirable, and this was afforded by reaction of lysozyme with phenylglyoxal (PG-Lysoz). In PG-Lysoz, one arginine residue was modified and it was located at position 61. The derivative had identical optical rotatory dispersion and circular dichroism parameters with those of lysozyme. However, minor conformational changes were present, and these were only revealed by the slight increase in disulfide reducibility (0·26 bonds) and by a small increase in susceptibility to tryptic attack (from 0·16 bonds in lysozyme to 0·97 bonds in PG-lysozyme). The enzymic activity of PG-Lysoz was suppressed slightly (to 83 per cent) but the pH optimum was unaltered. With antisera to lysozyme, PG-Lysoz showed equal antigenic reactivity to that of lysozyme. Conversely, with antisera to PG-Lysoz, the reactions of lysozyme and PG-Lysoz were identical. Also, the two proteins had equal reactivities with antisera to CHD-Lysoz I (58–60 per cent relative to homologous antigen). It was, therefore concluded that arginine-61 is not located in an antigenic site of lysozyme. Also, the findings demonstrate that not all conformational changes will influence antigenic reactivity. The change in the latter will be dependent on the protein and on the nature of the conformational reorganization.