DENATURATION AND INACTIVATION OF ENZYME PROTEINS

I. BACTERIAL PROTEINASE METHOD FOR THE DETERMINATION OF THE RATIO OF DENATURATION OF GLOBULAR PROTEINS

抄録

It was confirmed that native enzyme proteins, crystalline bacterial and Taka-α-amylase, were not digested by bacterial proteinase, trypsin or chymotrypsin, but the denatured proteins were rapidly digested with such proteinases. With trypsin or chymotrypsin, however, the denatured proteins were difficult to be digested to the state completely soluble in 0.4M TCA, in which undigested proteins-native proteins-were completely insoluble. With the bacterial proteinases, the digestion products soluble in TCA rapidly reached a constant value measured by ultraviolet absorption or Folin method. The susceptibility of denatured proteins to the proteinase was practically the same whether the denaturation occurred partially or completely. The same concentration of denatured proteins which was produced through various ways gave the same amount of digestion product by the action of bacterial proteinase. Based on the above facts, a convenient and accurate method for determining the “ratio of denaturation”-quantitative determi-nation of denatured and native states-of partially denatured proteins has been presented, in which bacterial proteinase was used to digest a denatured protein and the digestion product was measured by ultraviolet absorption and Folin method after separating from undigested proteins with TCA. When this method was used for partially denatured Taka-α- amylase, the “RD” agreed well with the “ratio of inactivation.” Comparison of this method with other methods for determining the “RD” of the protein was also discussed.
We are most grateful to Nagase Ltd. and Sankvo Ltd. for their gifts of source materials of enzyme.