Nitration of pancreatic trypsin inhibitor with tetranitromethane affords a mono-and a disubstituted derivative which was shown by sequential analysis to contain nitrotyrosine in position 10 and in positions 10 and 21, respectively. Both retain full trypsin inhibitor activity. Measurements of optical rotatory dispersion in the 230 nm region indicate that the secondary structure of the protein is unaffected by nitration. The nitrated derivatives exhibit a conformation-dependent side-chain Cotton effect in the region of nitroaromatic absorption. No such Cotton effect is shown by a nitrotyrosine-containing pentadecapeptide isolated from the tryptic digest from the nitrated and oxidized inhibitor. We have earlier shown [a] that the optical rotatory dispersion curve of pancreatic trypsin inhibitor shows a Cotton effect at 278 nm due to the tyrosine residues and that this effect is strongly dependent on conformation. It therefore appeared of interest to modify the tyrosine residues and to study the effect of such modification on the inhibitory activity and optical rotatory dispersion behaviour of pancreatic trypsin inhibitor. The recently introduced tetranitromethane reagent [9-111 has accordingly been used to nitrate pancreatic trypsin inhibitor and the properties of the nitrated product have been investigated. MATERIAL AND METHODSPancreatic trypsin inhibitor was prepared and characterized as described in the preceding paper [4].Non-Standard Abbreviation. Dansyl, I-dimethylaminonaphthalene-5-sulfonyl. Enzyme. Trypsin (EC 3.4.4.4).Trypsin used for the digestion was a crystalline preparation obtained from LBFiva (Prague) and was recrystallized three times with magnesium sulfate. Its residual chymotryptic activity was inhibited by reaction with l-chloro-4-phenyl-3-tosylamino-2-bu-The activity of trypsin was determined according to Nagel et al.[13] from the increase of absorbance at 405 nm due to the p-nitraniline liberated from benzoyl-DL-arginine p-nitranilide.Pancreatic trypsin inhibitor activity was determined by a difference method. The solution to be assayed (50 pl) was incubated with 100 pl of 0.1 M Tris-HC1 buffer, pH 7.8, and 250 pl of 0.001 N HC1 containing 40 pg of trypsin at 25" for 1, 5, I0 or 15 minutes and the activity of the remaining trypsin was determined as above.Protein concentrations were measured spectrophotometrically using A&ilo = 17.24 for trypsin s o htions and AiAlo = 8.33 for solutions of the inhibitor.Ultraviolet absorption spectra and optical rotatory dispersion curves were measured on a Jasco ORD/UV-5 recording spectrophotometer and spectropolarimeter. The absorption spectra were recorded in 1 cm cells. Solutions in 0.05 M sodium acetate (pH 4.6) and in 0.1 M Tris-HC1 buffer (pH 7.8) were prepared from weighed amounts of the dry protein (about 0.04 g/100 ml). Optical rotatory dispersion measurements were performed with the same solutions in cells of 1.0, 0.1, and 0.01 dm light path. For some optical rotatory dispersion measurements anhydrous trifluoroacetic acid served as the solvent. The so...

Guanylyl‐3′‐5′‐uridine (GpU) and seventeen GpU analogues modified mostly on the uridine moiety have been investigated by circular dichroism (CD). The following conclusions can be drawn from this investigation. At room temperature and neutral pH GpU is very little, if at all stacked. A perpendicular structure with guanosine in syn and uridine with ϕCN∼+75°± 15°C stabilized by a hydrogen bond between the amino group of guanosine and the C‐4 keto group of uridine is proposed. In this structure C‐5 and C‐6 would be hidden by the guanosine base. At low temperature or low pH stacking occurs. The structure is possibly the same under these conditions and may involve guanosine in syn and uridine in anti. Substitution at C‐5 of uridine inhibits the formation of the neutral perpendicular structure. Seven C‐5 substituted GpU analogues all show CD spectra close to those of GpU at low temperature or low pH. Substitution on C‐6 of uridine destabilizes the formation of all these structures.

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A mathematical model has been developed which uses the theory of Hilbert spaces and the modified method of the principal components of the factor analysis to the determination of the minimum number of subspectra sufficient to describe a set of experimental CD curves of a series of structurally related compounds. The use of this method has been demonstrated on the CD spectra of nine cyclodipeptides containing (besides L-proline) glycine, L or D alanine, L or D valine, L or D leucine, and L or D tert-leucine, measured in acetonitrile and 2,2,2-trifluoroethanol. Relation is discussed between the calculated subspectra and the structural characteristics of the measured systems.

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