The state of tyrosine in egg albumin and in insulin as determined by spectrophotometric titration

Crammer, J. L.; Neuberger, A.

doi:10.1042/bj0370302

Cited by 267 publications

(75 citation statements)

References 17 publications

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“…In proteins where tryptophan accounts for the major part of the near-ultrayiolet absorption its concentration can be determined more precisely than that of tyrosine by absorption measurements. An independent method of tyrosine analysis, however, is available based on the major enhancement and shift toward longer wavelengths of its absorption spectrum that occurs with ionization (Crammer and Neuberger, 1943). Since the indole moiety does not have any ionizable groups its spectrum should be unaffected by the change in pH required to titrate the phenolic moiety.…”

Section: Resultsmentioning

confidence: 99%

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*

Edelhoch¹

1967

Biochemistry

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A rapid method for the determination of tryptophan in proteins is presented. It is based on absorbance measurements at 288 and 280 mp of the protein dissolved in 6 M guanidine hydrochloride. Blocked tryptophanyl (N-acetyl-L-tryptophanamide) and tyrosyl (glycyl-L-tyrosylglycine) compounds were selected as C urrent methods of protein amino acid analysis do not give quantitative values for tryptophan and consequently the amino acid compositions, which are otherwise complete, fail to report tryptophan values. The principal reason for this situation is that the standard procedure of protein hydrolysis in strong acid results in the destruction of tryptophan (Hill, 1965). Therefore a second procedure is required to measure tryptophan. Alkaline hydrolysis is less destructive but does not give quantitative recoveries generally (Spies and Chambers, 1949). Enzymatic hydrolysis of proteins can give quantitative yields of tryptophan but this method may not be generally valid (Hill and Schmidt, 1962).The hydrolytic problem can be circumvented by measuring tryptophan in the intact protein. A chemical method has been developed which has not been exploited adequately Chambers, 1948, 1949). On the other hand, considerable effort has been expended in developing absorption spectroscopic procedures to measure tryptophan and tyrosine in unhydrolyzed proteins. Holiday (1936) and Goodwin and Morton (1946) have measured the absorption of proteins in 0.1 M NaOH and computed their tryptophan and tyrosine contents based on comparison with the absorption of the two amino acids. A modification of these techniques has been presented by Bencze and Schmid (1957). The preceding three methods do not give quantitative results. The behavior of the chromophores has not been normalized and the two models, i.e., tryptophan and tyrosine, are not completely adequate. A procedure is suggested in this report which strongly reduces or eliminates these interactions, normalizes their absorption, and consequently permits a more precise analysis of tryptophan and tyrosine in proteins.

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Section: Resultsmentioning

confidence: 99%

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*

Edelhoch¹

1967

Biochemistry

3,600

1,728

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“…The absorption of ionized tyrosine is enhanced and shifted to longer wavelengths as compared to protonated tyrosine [18], giving rise to a difference spectrum with a maximum at 295 nm. Fig.…”

Section: Refolding Of Rnuse a Monitored By Tyrosine Ionizationmentioning

confidence: 99%

A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Schmid

Blaschek

1981

European Journal of Biochemistry

117

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A folded intermediate Iy has been detected in the slow refolding reaction of ribonuclease A, US -+ N, where Us is an unfolded species which contains non-native isomers of X-Pro peptide bonds and N is the native enzyme. IN has been characterized previously as a structured folding intermediate : it shows a tyrosine absorbance like native ribonuclease, but it still contains at least one non-native proline peptide bond and its tyrosine fluorescence differs from the native enzyme.Here we have used three probes to characterize further the structure of I N in comparison to native ribonuclease A.(1) The accessibility of the tyrosine hydroxyl groups to ionization has been investigated in IN. (2) The binding of the specific inhibitor cytidine 2'-phosphate (2'CMP) to IN was measured. (3) The regain of enzymatic activity during folding was monitored to decide whether IN is an enzymatically active intermediate.The results indicate that I N possesses a compact folded structure which shields part of the tyrosine residues from the aqueous environment like native ribonuclease A. The active-site region of IN has folded to the native conformation. IN binds 2'CMP as well as native ribonuclease A does and it shows enzymatic activity similar to the native enzyme.We conclude that unfolded molecules, which have only proline-93 in the non-native trans configuration, can refold to the native-like intermediate IN before proline isomerization takes place. Proline-93 is located far away from the active-site region in an external bend and therefore does not block folding. It isomerizes in the folded molecule to the native cis configuration.Unfolded RNase A consists of 20% fast-folding species, UF, and 80% slow-folding species, Us, which coexist in a slow UF -+=-Us equilibrium [I -31. US folds slowly because it contains non-native proline isomers [4-61. The kinetic pathway of refolding of US depends on the final folding conditions ; under strongly native conditions kinetic intermediates have been observed in the course of the US --f N reaction. An early hydrogen-bonded intermediate was identified by measuring competition between amide proton exchange and refolding [7], and a native-like intermediate I N was detected by Cook et al.[8] via a comparison of the absorption changes during the Us + N reaction with the time course of proline isomerization during refolding of US. They concluded that IN is a folded intermediate which shows a tyrosine absorption like native RNase A and which is capable of binding 2'CMP, a specific inhibitor of RNase A. However, IN still contains at least one essential proline in a non-native configuration. Contrary to the changes in tyrosine absorption, the native tyrosine Dedicated to Professor Rainer Jaenicke on the occasion of his fiftieth birthday.Abhreviutions. RNase A, bovine pancreatic ribonuclease with disulfide bonds intact; guanidine . HCI, guanidine hydrochloride; Z'CMP, cytidine ;!'-phosphate; 2',3'CMP cytidine cyclic 2',3'-phosphate; T, time constant of a reaction or kinetic phase (reciprocal of the apparen...

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“…The action of urea (2, 3) involves a weakening of the association of adjacent polypeptide chains by competition of the urea for the hydrogen bonding affinity of the peptide linkage and the breaking of other hydrogen bonds, between side chains, and possibly also of other linkages in the protein molecule. On the basis of ultraviolet absorption studies at different pH values, Crammer and Neuberger (19) have recently concluded that in native ovalbumin the phenolic hydroxyl groups in the tyrosine residues are involved in hydrogen bonds of considerable strength. The importance of the bonds formed by these and other residues in maintaining protein structure and the susceptibility of these bonds to the various denaturing agents await further investigation.…”

Section: Discussionmentioning

confidence: 99%

Studies on the Denaturation of Antibody

Wright

1944

Journal of Experimental Medicine

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Although a great deal of information has been accumulated regarding the stability of antibody under various conditions and treatments (1), the action of urea and similar substances seems to have been largely neglected. Since the action of urea in denaturing proteins involves weakening the secondary bonds about the peptide linkages (2, 3) it is of interest to know the effect of urea on antibody. Pappenheimer, Lundgren, and Williams (4) report that diphtheria antitoxin is unaffected by "strong" urea solutions. Very recently Erickson and Neurath (5) have reported some experiments on the action of guanidine hydrochloride on horse antipneumococcus antibody.The present investigation of the stability of antibody in urea was undertaken because of the presumable importance of the results to the theory of antibody structure. Diphtheria antitoxin has been chosen as the antibody for study because the accurate neutralization methods available for its determination permit direct measurement of the fundamental property of antibody, its ability to combine with antigen. Materials and MethodsThe antitoxic globulin used was a commercial preparation obtained from horse plasma by ammonium sulfate fractionation between 32 and 50 per cent saturation. It contained 20 per cent protein, and had a potency of 3000 units of antitoxin per ml.; phenol was present as a preservative. Quantitative precipitation tests (6) showed that 18 per cent of the protein was precipitable with toxin. Electrophoretic analysis in sodium phosphate buffer of pH 7.8 and ionic strength 0.1 indicated that the globulin consisted mainly of the T component (7), pins a small amount of gamma globulin and traces of faster components. The toxin contained 42.5 Lf per ml. Both toxin and antitoxin were obtained from Sharp and Dohme, G1enolden, Pennsylvania, through the courtesy of Dr. Arnold Welch. Most of the antitoxin titrations were carried out by the Ri~mer intracutaneous method in rabbits (8). The accuracy of these titrations, as determined by the dilution intervals used, was better than 5 per cent in most of the determinations. However, in part A and in instances in which a large amount of inactivation occurred the error approached 10 per cent.

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The state of tyrosine in egg albumin and in insulin as determined by spectrophotometric titration

Cited by 267 publications

References 17 publications

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*

A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Studies on the Denaturation of Antibody

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The state of tyrosine in egg albumin and in insulin as determined by spectrophotometric titration

Cited by 267 publications

References 17 publications

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins*

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins*

A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Studies on the Denaturation of Antibody

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Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*

Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*