lnteraction of ribonuclease F1 with various guanine nucleotides as studied by differential absorption spectra

Abstract

lnteraction of various guanine and related nucleotides with ribonuclease Fl was studied by means of difference absorption spectra. Spectrophotometric titrations were carried out on a total of 16 nucleotides which were classined into three groups; (A)guanine and hypoxanthine nucleotides(6)，(B)8-substituted guanine nucleotides (6) and(C) dinucleotides (4)(the numbers of nucleotides are shown in parentheses). Computer-aided data processing enabled determination of the difference molar absorbance coefficient △Emax and the dissociation constant of the ribonuclease F1-nucleotide complex Kd for each nucleotide. Among group A nucleotides，guanosine 2'-phosphate showed the lowest Kd (the highest affinity) and inosine 2'-phosphate the highest Kd (the lowest affinity).The difference between their binding free energy changes was estimated to reach 2.3 kcal/mol. Among group B nucleotides，8-aminoguanosine 2'-phosphate exhibited the lowest Kd，which was even lower than that ofguanosine 2'-phosphate. The dinerence in their binding free energy changes was l.0 kcal/mol. Group C nucleotides had higher Kd than their common component，guanosine 3'-phosphate，suggesting destabilizing effect on complex formation by an adjacent nucleotide. A11 these results were similar but not completely the same for those reported on ribonuclease TI.