A Consideration on the Ultraviolet Spectrum of Ribonuclease A-Inhibitor Complex

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Abstract
1. A hypothesis which explains the difference spectrum between that of the RNase A [EC 2.7.7.16]-inhibitor complex and those of its constituents was proposed. This hypothesis is based on two assumptions, (i) On formation of the El-complex, the base moiety of the nucleotide comes in contact with the side chain of the peptide back bone of the enzyme, which has a higher refractive index than that of the surrounding medium and the spectrum of the base moiety shifts with the change of refractive index in its environment. (ii) The stacking or interaction of the aromatic ring of an amino acid side chain with the base moiety of the inhibitor causes some hypochromism in the spectrum of the El-complex. 2. The above assumptions were based on the following evidence. (i) The λmax of the El-complex was shifted slightly to a longer wavelength than that of the sum of those of E and I. (ii) In a solvent of high refractive index, such as 80 per cent ethyleneglycol or 40 per cent dimethylformamide, which has almost the same refractive index as that of the hydrophobic part of the protein molecule, the effect of environmental change (change in refractive index) was very small. In such a solvent, the difference spectrum of the El-complex was still observed and the λmax was at a slightly higher wavelength while the Δɛ was smaller than in aqueous solution. This small difference in Δɛ could be an indication of some kind of hypochromicity in the formation of the El-complex. 3. On the bases of the above hypothesis, the spectrum of a competitive inhibitor, such as C-2'-p or Up was modified artificially (to give 2 mμ red shift and 20 per cent hypochromicity for C-2'-p and 1.5 mμ red shift and 20 per cent hypochromicity for Up). The difference spectrum between the modified spectrum and that of the parent nucleotide was found to be qualitatively similar to the difference spectrum of the EI-complex. 4. The possible role of phenylalanine residues in RNase A was discussed on the basis of the above hypothesis.

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