Hiroo Horinishi

States of amino acid residues in proteins: V. Different reactivities with H2O2 of tryptophan residues in lysozyme, proteinases and zymogens

Abstract Hydrogen and organic peroxides were examined in a search for a reagent to differentiate between various states of tryptophan residues in proteins. Hydrogen peroxide in a solution of 0.5 M bicarbonate buffer (pH 8.1–9.4) containing 10% dioxane was found to possess a moderate oxidizing power to oxidize free and bound tryptophan residues at different concentrations of H2O2, and the strength of the tryptophan absorption band of the protein was lowered stepwise with increasing concentration. By the use of this reagent, tryptophan residues in several proteins were classified into various types with different oxidizabilities, and the moles, n, of each type per mole of proteine were determined; n=5 and 1 for lysozyme (EC 3.2.1.17), n=5, 1 and 1 for chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5), and n=2, 1 and 1 for trypsinogen and trypsin (EC 3.4.4.4.) which are arranged in the order of decreasing oxidizability. On the activation of chymotrypsinogen, the oxidizability of the most strongly bound residue decreased greatly whereas, on the activation of trypsinogen, the oxidizability of the secondly oxidized residue increased slightly. The structural rearrangement on the activation was discussed in terms of these changes in state of tryptophan as well as those of other amino acids determined previously. The enzymic activity of lysozyme an proteinases was also measured as a function of H2O2 concentration, and the results were correlated with the degree of oxidation of tryptophan residues. All of the tryptophan residues in denatured proteins were oxidized at a low concentration of H2O2, so that the reagent is applicable for the determination of the molar tryptophan content in protein.

Reactions of cyanuric halides with proteins I. Bound tyrosine residues of insulin and lysozyme as identified with cyanuric fluoride

Abstract The ultraviolet absorption band of tyrosine was found to be remarkably lowered and shifted toward shorter wavelengths by treatment with cyanuric fluoride, (CNF)3, whereas the bands of other amino acids were either unaffected by the treatment or affected below 295 mμ, the peak position in the difference spectrum of the ionization of tyrosine. By use of this phenomenon, the reactivities of the tyrosine residues of lysozyme and insulin with cyanuric fluoride were examined, and the following facts were established. Two of the three tyrosine residues in the lysozyme molecule react with cyanuric fluoride and the remaining one is non-reactive. Of the four tyrosine residues in the insulin molecule, two are reactive while the other two are non-reactive. Upon addition of alkali to an insulin solution, one of the two non-reactive residues is transformed rapidly and the other slowly into the reactive type. Of these two bound residues of the non-reactive type one is in the A the other in the B chains of insulin. The numbers and the positions of the bound residues of the same proteins, identified by other properties, are discussed.

Reactions of glyoxal with nucleic acids, nucleotides and their component bases

Abstract The reactions of glyoxal with nucleic acids, nucleotides and their bases were examined by measuring spectral changes. The spectra of all of the bases and nucleotides in RNA and DNA were changed appreciably by treatment with a high concentration of glyoxal. When treated with a low concentration of glyoxal, however, only guanine or guanylic acid underwent a specific spectral change. The reaction of glyoxal with this base or its nucleotide proceeded to completion within 30 min between pH 8.3 and 9.8 as estimated from the spectral change at room temperature. This phenomenon was applied for determination of the guanosine content of nucleic acids and the results agreed well with previous data obtained by chromatography. As judged from the spectral change, glyoxal did not react with native calf thymus DNA, while it reacted considerably with heat-denatured DNA. The spectral change obtained with denatured DNA was a composite of two qualitatively different changes, the change being due solely to the reaction between glyoxal and guanosine residues and the hyperchromic shift of the 260-mμ band, which may be a secondary effect of the reaction. From the analysis of these two effects, the degree of reaction of the guanosine residues in denatured DNA was estimated to be 72 %.

States of amino acid residues in proteins VIII. Tyrosine, histidine and tryptophan residues in chymotrypsin in the presence of substrate and in diisopropylphosphoryl-chymotrypsin

Summary Tyrosine, histidine and tryptophan residues in α-chymotrypsin (EC 3.4.4.5) in the presence and absence of benzoylglycine methyl ester and those in diisopropylphosphoryl-chymotrypsin were analyzed by measuring their reactivities with cyanuric fluoride, diazonium-1-H-tetrazole and H2O2-dioxane, respectively, which are the reagents to differentiate between various states of these residues in protein. By the diisopropylphosphorylation of α-chymotrypsin, two of the total seven tryptophan residues, both of the two histidine residues and one of the four tyrosine residues in the molecule were bound more strongly or burried more deeply. These effects of phosphorylation were discussed from two different view points ; direct effects of the diisopropylphosphoryl group introduced into the specific serine residue and indirect effects of the conformational change induced by the phosphorylation. The addition of benzoylglycine methyl ester to a chymotrypsin solution lowered the reactivity of one of the tyrosine residues with cyanuric fluoride to the same extent as in the phosphorylation, but the histidine and tryptophan residues were unaffected by the presence of substrate. It was inferred from this highly specific effect of substrate that the tyrosine residue is masked by the substrate molecule and is involved in the specific binding of substrate to the enzyme. The activity measured with the same synthetic substrate dropped with pK = 9.6 slightly lower than pK = 10.2 obtained for the ionization of one of the tyrosine residues, and 2 moles of OH− were involved in the inactivation.

States of amino acid residues in proteins. XVIII. A revised way of using diazonium-i-H-tetrazole for reactivity examination of histidine and tyrosine residues

Abstract Diazonium- i -H-tetrazole explored previously for discrimination of various states of histidine residues in proteins affords spectrally different bisazo derivatives of histidine and tyrosine, having a visible absorption band at 480 and 548 mμ, respectively. The overlapping effects of monoazotyrosine and bisazotyrosine bands on the bisazohistidine band was neglected in the previous method, and this approximation was pointed out later to include considerable errors. Corrections were made in the present study, taking account of all the mutual overlapping effects of the visible bands of bisazohistidine, monoazotyrosine and bisazotyrosine, and revision was made in the corrected method to determine the concentrations of these three derivatives of histidine and tyrosine residues. Application of the revised method to insulin revealed the following facts which indicate that the overlapping effects are properly corrected in the revised photometric method. One of the total two histidine residues in the insulin molecule is biscoupled, and the other remains as the monoazo derivative. Three of the four tyrosine residues are biscoupled, and the other one residue remains as the monoazo derivative. The two α-amino groups at A and B peptide termini and the e-amino group of B29 Lys are modified with the rates in the order, A1 Gly > B1 Phe > B29 Lys.

States of amino acid residues in proteins: VI. Heme-linked histidine residues in cytochrome c as determined with diazonium-1-H-tetrazole

Abstract Diazonium-1-H-tetrazole(DHT), a new coupling reagent for histidine residues, was reacted with horse heart and bakers yeast cytochrome c to differentiate between free and heme-linked histidine residues. Coupling to the histidine residues in horse heart cytochrome c proceeds in three steps with increasing DHT concentration, such that each of the three histidine residues in the molecule is coupled at a different DHT concentration. Coupling to the four histidine residues in the yeast cytochrome c molecule also proceeds in three steps. Two of the residues are coupled in the first step, and each of the remaining two residues at higher DHT concentrations in the second and in the third steps, respectively. The Soret band of these cytochromes does not change in the first step when the most reactive histidine residue(s) is coupled, but changes stepwise in parallel with the second and the third stages of coupling. Reduction of ferricytochrome c appreciably decreases the reactivities toward DHT of the two residues responsible for the second and the third steps, but affects slightly or not at all the reactivity of the most reactive residue(s). Dihistidyl coordination to the iron atom of heme is proposed from these results. The two iron-linked histidine residues were located at positions 18 and 26 in the amino acid sequence by digestions with trypsin and with Nagarse of DHT-treated horse heart cytochrome c , in which only the most reactive histidine residue was modified with DHT.

States of amino acid residues in proteins: IX. Histidine residues in ribonuclease in the presence of cytidine- and uridine-3′-phosphates as observed with diazonium-1-H-tetrazole

Abstract Diazonium-1-H-tetrazole (DHT), a new coupling reagent for histidine residues, was reacted with bovine pancreatic ribonuclease (RNase) in the presence and absence of nucleotides; cytidine-3′-phosphate (CMP), uridine-3′-phosphate(UMP), adenosine-3′-phosphate (AMP), and guanosine-3′-phosphate (GMP). Coupling to the four histidine residues in the native RNase molecule in the absence of nucleotide proceeds in four steps with increasing DHT concentration, such that each of these four residues is coupled at a different DHT concentration. The reactivities were affected by the presence of CMP or UMP, a product of the RNase-catalyzed reaction, whereas AMP or GMP, which are not products of the reaction, show no effect on the reactivities. The effect of CMP is different from that of UMP; CMP enhances the reactivity of a residue which is the third to be coupled in the absence of nucleotide, while UMP enhances the reactivity of a residue which is the second or fourth to be coupled. The possible participation in the enzymic activity of these two histidine residues with different reactivities and their different responses to nucleotides were thus demonstrated.