The State of Tyrosine and Phenylalanine Residues in Proteins Analyzed by Fourth-Derivative Spectrophotometry. Histone H1 and Ribonuclease A

Abstract

The analysis of the absorption spectra of model compounds of tyrosine and phenylalanine residues by means of fourth-derivative spectrophotometry is able to separate the contribution of the two chromophores, thus allowing the study of each one. Fourth-derivative analysis resolves the two main vibrational bands of tyrosine, giving rise to two peaks which are sensitive to changes in the environment of the phenolic ring. The parameters obtained from the fourth-derivative spectra were found to depend on the strength of the hydrogen bonds formed by the O H group of tyrosine, as well as on the heterogeneity of tyrosine environments. It is also shown that the fourth-derivative tyrosine peaks are not perturbed by broad bands, such as that arising from ionized tyrosine chromophores. The peaks arising from the phenylalanine model, although less sensitive than those of tyrosine, were found to depend on the polarity of the environment. As a check of the method, it is applied to the study of tyrosine and phenylalanine residues of calf thymus histone H1 and bovine pancreatic ribonuclease A.The near-ultraviolet absorption spectrum of aromatic residues in proteins contains a vast amount of information that is masked by the large width of the absorption bands as compared with the small changes that occur on varying the local environment of the chromophores [I, 21. Several methods of difference spectrophotometry and derivative spectrophotometry have been developed in an effort to obtain as much information as possible from the absorption spectra [2-41, but at present the majority of such information remains unresolved.This communication reports the application of the fourthderivative method to the analysis of the absorption spectra of tyrosine and phenylalanine residues. The main advantages of using fourth-derivative spectroscopy have been pointed out by Butler [5] : (a) the maxima of the fourth-derivative curves are located at nearly the same position as in the original spectrum ; (b) the fourth-derivative analysis can reveal the presence of constituent bands that are not apparent in the original spectrum ; (c) the amplitude of the fourth-derivative curve approaches an inverse fourth-power dependence on the half-width of the original band, thus selecting the narrow bands.We first applied this method to model compounds of tyrosine and phenylalanine residues in different solvents, in order to simulate the various types of environments of these residues in proteins. The conclusions obtained were then applied to the study of conformational changes of calf thymus histone H I and ribonuclease A from bovine pancreas. These proteins are well-suited to be used as a test for the fourthderivative method. The H1 molecule contains only one tyrosine residue and one phenylalanine residue, and RNase A contains six tyrosines and three phenylalanines. Both proteins MATERIALS A N D METHODSThe following solvents of spectroscopic quality were purchased from Xpectrix (Barcelona, Spain) : acetonitrile,

Fourth‐Derivative Spectrophotometry Analysis of Tryptophan Environment in Proteins

“…I shows the absorption and the fourth-derivative spectra of AcTrpNH, in water, pH 7.0. The general appearance of the fourth-derivative curve is similar to that obtained for the corresponding tyrosine model [4]. The absorption spectrum of AcTrpNHz in this spectral region is the sum of sharp 1Lb transitions superimposed with broad structureless 'La bands [8].…”

“…I., and 12 are the wavelengths of the minimum and the maximum of the long-wavelength peak, respectively. The nomenclature used in this paper for the wavelengths and the peak intensities is at variance with that of the previous paper [4] Solvent R Table 1). The marked differences between the fourth-derivative results for AcTyrOEt [4] and AcTrpNHz deserve some comments.…”

“…Reduction was accomplished by a similar procedure, using sodium dithionite. Fourth-derivative spectra were taken on a Perkin-Elmer model 320 spectrophotometer, using a derivative interval of 4 nm, a slit width of 1 nm, a time constant of 1 s, and a scan rate of 30 nm/min [4]. The samples were prepared and run at least in duplicate.…”

“…The fourth-derivative operation is a general method for resolving overlapping bands, provided that there exists sufficient difference between the bandwidths [3]. We have previously applied this method to the study of Tyr and Phe residues, and have shown that it is able to separate the contribution of these two residues, thus allowing their independent study [4]. The progressive availability of microcomputer-controlled spectrophotometers ensures that to obtain the fourth-derivative spectra is now a straightforward matter in'most laboratories.…”

Chloroplast fructose‐1,6‐bisphosphatase: Modification of non‐covalent interactions promote the activation by chimeric Escherichia coli thioredoxins

“…The analysis of the absorption spectra by means of fourthderivative spectrophotometry resolves the contribution of tyrosine and tryptophan residues, thus allowing the study of their environments [21,22]. In a previous publication [19], we detailed four different spectra of chloroplast fructose-l,6-bisphosphatase obtained by this technique: (i) the native form, and the species incubated with (ii) fructose 1,6-bisphosphate and Ca 2+, (iii) fructose 1,6-bisphosphate, Ca 2+, and trichloroacetate, and (iv) trichloroacetate.…”