Radiation chemistry of proteins: II. Enzymic activity and deuterium exchange properties of lysozyme and α-chymotrypsin

Abstract

Chromatographically homogeneous egg-white lysozyme and crystalline α-chymotrypsin were irradiated in a Cs 137 source with γ-rays, and the resulting samples were subjected to amino acid analyses, chromatographic examination, and deuterium exchange measurements. After a 10.7 Mrad dose 3 one residue each of serine and tryptophan are lost per mole of α-chymotrypsin while in lysozyme, after a 26.6 Mrad dose 3 3 At these radiation doses the enzymic activity of a lysozyme or α-chymotrypsin sample is reduced by 37%. This level of irradiation was deliberately chosen for detailed study of possible amino acid residue destruction and chromatographic separation of radiation products. At this dose level one may expect to observe chemical changes predominately associated with loss of enzymic activity (30, 31). At higher dose levels, approaching complete enzymic activation in the sample, one would expect repeated ionizations and consequent radiation damage in some molecules that had already experienced a damaging radiation event. Thus, it would be difficult to ascertain whether loss of enzymic activity resulted from a chemical change resulting from a single, supposedly lethal (30, 31), or a second or third ionization within or near the enzyme molecule. , no significant loss in any amino acid residue is observed. From irradiated α-chymotrypsin only one discrete active component can be demonstrated chromatographically. Chromatographic examination of the phosphate soluble material in irradiated lysozyme reveals five active and one inactive components. In α-chymotrypsin hard-to-exchange-amide (peptide) hydrogens are lost at about the same rate as enzymic activity as a function of radiation dose. In lysozyme, irradiation disrupts conformation so that hard-to-exchange-amide (peptide) hydrogens are lost at least twice as rapidly as enzymic activity. These results indicate that enzymic activity in α-chymotrypsin is closely dependent on native molecular conformation whereas only about half the native conformation of lysozyme is essential to enzymic activity.