NMR STUDY ON THE MAJOR COMPONENTS IN HEMATOPORPHYRIN DERIVATIVE YHPD

The hematoporphyrin derivative YHPD, a China-made product, has been clinically used in photodynamic therapy of tumors as a good photosensitizing drug. The NMR study on the structure of its major components is reported here. In terms of high performance liquid chromatography (HPLC) four major components A, B, C and D were isolated. The NMR results showed that the component A is O-acetylhematoporphyrin, B and C are two isomers of vinyldeuteroporphyrin. The spectra of 2-dimensional homonuclear correlation NMR, 2-dimensional NOE (nuclear overhauser enhancement), ~(13)C-NMR and off-resonance as well as FAB (fast atom bombarding) mass spectrum of component D indicate that it is a protoporphyrin dimer linked by carbon-carbon bond. This finding may providea chemical basis for understanding the difference in biological activity between YHPD and other foreign commercial HPD, as well as the composition of clincally used alkali-treated HPD and its effective component.     

NMR study on the major components in hematoporphyrin derivative YHPD

The hematoporphyrin derivative YHPD, a China-made product, has been clinically used in photodynamic therapy of tumors as a good photosensitizing drug. The NMR study on the structure of its major components is reported here. In terms of high performance liquid chromatography (HPLC) four major components A, B, C and D were isolated. The NMR results showed that the component A is O-acetylhematoporphyrin, B and C are two isomers of vinyldeuteroporphyrin. The spectra of 2-dimensional homonuclear correlation NMR, 2-dimensional NOE (nuclear overhauser enhancement), 13C-NMR and off-resonance as well as FAB (fast atom bombarding) mass spectrum of component D indicate that it is a protoporphyrin dimer linked by carbon-carbon bond. This finding may provide a chemical basis for understanding the difference in biological activity between YHPD and other foreign commercial HPD, as well as the composition of clinically used alkali-treated HPD and its effective component.     

Intramolecular quenching of tryptophan phosphorescence in short peptides and proteins

The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys > His+ > Tyr > Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.     

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.     

Triplet state sublevel spectroscopy applied to proteins

Triplet state sublevel spectroscopy using optical detection of magnetic resonance (ODMR) in zero magnetic field can be successfully employed to study (i) the environment of tryptophan (Trp) residues in a protein by observing the position and structure of phosphorescence spectra, zero field ODMR transitions and triplet state sublevel kinetics, (ii) the energy transfer among Trp residues, and (iii) whether any cysteine (Cys) residue is within van der Waals distance of any Trp residue by studying the complex of the protein with methylmercury(II) iodide (CH₃HgI) which binds to Cys residues. These studies are particularly important where crystal structure study is not possible. Study of the S₁ state often gives ambiguous results since fluorescence is always broad and shows multi-exponential decay. Our results on bacteriophage lysozyme T4 which contains three Trp residues at positions 126, 138 and 158 are presented. Measurements were facilitated by the use of a mutated enzyme containing one or two Trp-Tyr substitutions. The results indicate that (i) Trp 126 and 158 are solvent exposed, whereas Trp 138 is buried in a hydrophobic environment, (ii)S ↔S non-radiative energy transfer takes place predominantly from Trp 126 to Trp 158, and (iii) only Trp-158 undergoes a heavy atom perturbation, which affects selectively the z-sublevel (z is an out-of-plane axis of the indole plane) as a result of CH₃HgI binding to nearby Cys 97. We suggest that the Hg atom is located on the z-axis of Trp 158 in the complex. This interpretation is based on our investigations on the effect of orientation of heavy atom perturbers in some naphthalene-crown ether metal ion complexes.     

Electrochemical oxidation of thrombin on carbon screen printed electrodes

The electrochemical oxidation of thrombin on the surface of carbon screen printed electrodes was studied. The electrochemical activity of thrombin was predicted, using bioinformation analysis, based on the data about the electrochemical properties of amino acids. The number of potentially electroactive amino acid residues, namely, tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), histidine (His), methionine (Met), and cystine (Cys-Cys) located on the protein surface and orientated by their electroactive groups toward the electrode surface, i.e., accessible for electrochemical oxidation was calculated. The theoretical data were confirmed experimentally by cyclic and square-wave voltammetry. The available data on the protein structure allowed us to attribute the recorded electrochemical signals of thrombin oxidation to certain types of amino acid residue: the oxidation peak with a potential maximum at 0.7–0.8 V (vs. Ag/AgCl) was attributed to the oxidation of the Trp and Tyr residues; the wave in the range 1.0–1.2 V, to the oxidation of His; and the wave at 1.2–1.5 V, to the oxidation of Met and Cys-Cys. The electroanalysis based on the oxidation peak of the Tyr and Trp amino acid residues allowed to detect thrombin up to the concentration of 10^–7 M. The suggested strategy for predicting the electrochemical activity can be used for investigating the properties of many other proteins and peptides and serve as a basis for their quantitative determination when developing various sensor and biosensor devices.     

三维同步偏振荧光光谱研究蛋白质溶液变性时氨基酸残基的发光行为

将偏振技术、同步技术与三维技术结合起来的三维同步偏振荧光光谱(TDSPS)能分辨蛋白质溶液中的色氨酸(Trp)和酪氨酸(Tyr)残基,具有同步光谱分辨率高、三维技术信息丰富的优点．本文用TDSPS表征牛血清白蛋白(BSA)、人血清白蛋白(HSA)受各种因素影响(酸效应、碱效应、盐效应、猝灭剂等)时Trp,Tyr残基荧光光谱的变化,用于区分HSA和BSA．     

A new way to understand quaternary structure changes of hemoglobin upon ligand binding on the basis of UV-resonance Raman evaluation of intersubunit interactions

The single residue vibrational spectra of tryptophan (Trp) and tyrosine (Tyr) residues in human adult hemoglobin (HbA), which play important roles in cooperative oxygen binding, were determined for the deoxy and CO-bound forms by applying UV resonance Raman spectroscopy to various variant Hbs. It was found that Trpβ37, Tyrα42, Tyrα140, and Tyrβ145 at the α(1)-β(2) subunit interface underwent transitions between two contact states (named as T and R) upon ligand binding, while Trpα14, Trpβ15, and Tyrβ35 displayed little changes. The corresponding spectral changes were identified only for the α(2)β(2) tetramer, but not the isolated α and β chains in the oligomeric forms, and therefore were exclusively attributed to a quaternary structure change. Ligand binding as well as allosteric effectors and pH altered only the number of the T-contacted Tyr and Trp residues without varying the two contact states themselves. A new method to semiquantitatively evaluate the amount of T-contacted Tyr and Trp residues in a given liganded form is here proposed, and with it a quaternary structure was determined for various symmetrically half-liganded forms obtained with ligand-hybrid, metal-hybrid, and valency-hybrid Hbs. It was found that ligand binding to the α or β subunits yielded different subunit contacts and that the contact changes of the Trp and Tyr residues were not always concerted. The contact changes at the α(1)-β(2) (α(2)-β(1)) interface are correlated with the proximal strain exerted on the Fe-His(F8) bond, which is noted to be much larger in the α than β subunits in the α(2)β(2) tetramer.     

Two-dimensional fluorescence correlation spectroscopy IV: resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.     

Site‐Selective Cysteine–Cyclooctyne Conjugation

We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water.     

Electrochemical determination of L-Tryptophan, L-Tyrosine and L-Cysteine using electrospun carbon nanofibers modified electrode

A novel and simple method for the direct and quantitative determination of L-tryptophan (Trp), L-tyrosine (Tyr) and L-cysteine (Cys) was proposed in this work. Carbon nanofibers (CNFs), made by electrospinning technique, were used to modify carbon paste electrode (CPE) without any treatment to study the electrochemical behaviors of the three amino acids using cyclic voltammetry (CV) and constant potential amperometric method. The results demonstrated that the CNFs modified carbon paste electrode (CNF-CPE) exhibited high electrocatalytic activity and good analytical performance towards the oxidation of the three amino acids. The linear ranges of Trp, Tyr and Cys were 0.1-119, 0.2-107 and 0.15-64 μM with correlation coefficients of 0.9994, 0.9985 and 0.9996, respectively. All the detection limits of the analytes were 0.1 μM (S/N = 3). In addition, the CNF-CPE displayed good reproducibility, high sensitivity and good selectivity towards the determination of the amino acids, making it suitable for the determination of Trp, Tyr and Cys in clinical and medicine.     

Radical-cationic gaseous amino acids: a theoretical study

Three major forms of gaseous radical-cationic amino acids (RCAAs), keto (COOH), enolic (C(OH)OH), and zwitterionic (COO(-)), as well as their tautomers, are examined for aliphatic Ala(.+), Pro(.+), and Ser(.+), sulfur-containing Cys(.+), aromatic Trp(.+), Tyr(.+), and Phe(.+), and basic His(.+). The hybrid B3LYP exchange-correlation functional with various basis sets along with the highly correlated CCSD(T) method is used. For all RCAAs considered, the main stabilizing factor is spin delocalization; for His(.+), protonation of the basic side chain is equally important. Minor stabilizing factors are hydrogen bonding and 3e-2c interactions. An efficient spin delocalization along the N-C(alpha)-C(O-)O moiety occurs upon H-transfer from C(alpha) to the carboxylic group to yield the captodative enolic form, which is the lowest-energy isomer for Ala(.+), Pro(.+), Ser(.+), Cys(.+), Tyr(.+), and Phe(.+). This H-transfer occurs in a single step as a 1,3-shift through the sigma-system. For His(.+), the lowest-energy isomer is formed upon H-transfer from C(alpha) to the basic side chain, which results in a keto form, with spin delocalized along the N-C(alpha)-C=O fragment. Trp(.+) is the only RCAA that favors spin delocalization over an aromatic system given the low ionization energy of indole. The lowest-energy isomer of Trp(.+) is a keto form, with no H-transfer.     

A theoretical study on the quenching mechanisms of triplet state riboflavin by tryptophan and tyrosine

The reactions of tryptophan (Trp) and tyrosine (Tyr) with endogenous photosensitizer riboflavin (RF) have gained much interest for their crucial roles in various photobiological processes. In this paper, the quenching mechanisms of triplet state RF by Trp and Tyr have been explored employing density functional theory calculations. It is revealed that the H-atom transfer reaction from Trp and Tyr to triplet state RF is more favorable on thermodynamic grounds compared with direct energy transfer or direct electron transfer pathways. During the photosensitization, RF can photogenerate various reactive oxygen species (ROS) as intermediates, while the present study provides some deeper insights into the photosensitizing behaviors of triplet state RF by reacting directly with Trp and Tyr.     

Comparison of various types of hydrogen bonds involving aromatic amino acids

Ab initio calculations are used to compare the abilities of the aromatic groups of the Phe, Tyr, Trp, and His amino acids (modeled respectively by benzene, phenol, indole, and imidazole) to form H-bonds of three different types. Strongest of all are the conventional H-bonds (e.g., OH..O and OH..N). His forms the strongest such H-bond, followed by Tyr, and then by Trp. Whereas OH..phi bonds formed by the approach of a proton donor to the pi electron cloud above the aromatic system are somewhat weaker, they nonetheless represent an important class of stabilizing interactions. The strengths of H-bonds in this category follow the trend Trp > His > Tyr approximately Phe. CH.O interactions are weaker still, and only those involving His and Trp are strong enough to make significant contributions to protein structure. A protonated residue such as HisH(+) makes for a very powerful proton donor, such that even its CH..O H-bonds are stronger than the conventional H-bonds formed by neutral groups.     

Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein‐derived cofactor. Reported herein is the discovery and elucidation of a Cys‐Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C?S) bond. By genetically incorporating an unnatural amino acid, 3,5‐difluoro‐tyrosine (F₂‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and ¹⁹F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low‐resolution techniques. Finally, a new sequence motif, C‐X‐Y‐Y(F), is proposed for identifying the Cys‐Tyr crosslink.     

Diethylpyrocarbonate Labeling for the Structural Analysis of Proteins: Label Scrambling in Solution and How to Avoid It

Covalent labeling along with mass spectrometry is a method that is increasingly used to study protein structure. Recently, it has been shown that diethylpyrocarbonate (DEPC) is a powerful labeling reagent because it can modify up to 30% of the residues in the average protein, including the N-terminus, His, Lys, Tyr, Ser, Thr, and Cys residues. We recently discovered, however, that Cys residues that form disulfide bonds appear to be modified by DEPC as well. In this work, we demonstrate that disulfide linked Cys residues are not actually reactive with DEPC but, instead, once reduced, free Cys residues can capture a carbethoxy group from other modified amino acids via a solution-phase reaction that can occur during the protein digestion step. This “scrambling” of carbethoxy groups decreases the amount of modification observed at other residues and can potentially provide incorrect protein structural information. Fortunately, label scrambling can be completely avoided by alkylating the free thiols after disulfide reduction.     

Aromatic Cluster Sensor of Protein Folding: Near‐UV Electronic Circular Dichroism Bands Assigned to Fold Compactness

Both far‐ and near‐UV electronic circular dichroism (ECD) spectra have bands sensitive to thermal unfolding of Trp and Tyr residues containing proteins. Beside spectral changes at 222 nm reporting secondary structural variations (far‐UV range), L_b bands (near‐UV range) are applicable as 3D‐fold sensors of protein's core structure. In this study we show that both L_b(Tyr) and L_b(Trp) ECD bands could be used as sensors of fold compactness. ECD is a relative method and thus requires NMR referencing and cross‐validation, also provided here. The ensemble of 204 ECD spectra of Trp‐cage miniproteins is analysed as a training set for “calibrating” Trp?Tyr folded systems of known NMR structure. While in the far‐UV ECD spectra changes are linear as a function of the temperature, near‐UV ECD data indicate a non‐linear and thus, cooperative unfolding mechanism of these proteins. Ensemble of ECD spectra deconvoluted gives both conformational weights and insight to a protein folding?unfolding mechanism. We found that the L_b²⁹³ band is reporting on the 3D‐structure compactness. In addition, the pure near‐UV ECD spectrum of the unfolded state is described here for the first time. Thus, ECD folding information now validated can be applied with confidence in a large thermal window (5≤T≤85 °C) compared to NMR for studying the unfolding of Trp?Tyr residue pairs. In conclusion, folding propensities of important proteins (RNA polymerase II, ubiquitin protein ligase, tryptase‐inhibitor etc.) can now be analysed with higher confidence.     

Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes

A series of twelve anionic, cationic, and neutral nickel(II) complexes have been synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity towards the different proteins based on the environment surrounding the Trp residue(s). Only small neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static quenching, with association constants ranging from 10(2) M(-1) (free Trp) to 10(10) M(-1) (lysozyme), indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme.     

ALKALINE QUENCHING OF BACTERIORHODOPSIN TRYPTOPHANYL FLUORESCENCE: EVIDENCE FOR AQUEOUS ACCESSIBILITY OR A HYDROGEN-BONDED CHAIN

Abstract— Comparison between Trp fluorescence yields of membrane-bound bacteriorhodopsin (BR) and retinylidene-free bacterioopsin (BO) is consistent with a model in which all eight Trp residues are active fiuorophores in the latter, while the emission of all but two residues in the former is lost by energy transfer to retinal. The visible chromophore of BR is progressively bleached with increasing pH. Up to pH 12 this bleaching is reversed on reneutralization; but above this the change is irreversible with the appearance of a new absorption band characteristic of free retinal. Emission yields for both proteins decrease with increasingly alkaline pH in a manner typical of energy transfer to weakly-fluorescent tyrosinate. The limiting yields, reached at a pH corresponding to that producing irreversible bleaching of the visible chromophore, agree with an integral value of one remaining active Trp fluorophore in BR and between one and two in BO and show that the bulk of Trp are within the 11 Å Förster energy-transfer distance of Tyr accessible to OH⁻. Current models of the native protein structure of BR arrange the polypeptide chain primarily in a bundle of seven helical segments with axes perpendicular to the lipid bilayer plane and with buried polar residues, including Trp and Tyr, located at intrahelical surfaces. An interpretation of the observed accessibility of buried Tyr to OH⁻ is that an aqueous region exists within the protein structure. Moreover, this putative aqueous region must be close to the retinylidene chromophore and thus may be associated with the light-driven ion transport system. The results are also compatible with energy transfer to internal Tyr residues which are connected via a chain of phenolate hydrogen bonds to a surface Tyr.     

Human milk lysozyme: unpublished data concerning the establishment of the complete primary structure; comparison with lysozymes of various origins

Details concerning the establishment of the complete primary structure of human milk lysozyme (previously published in a preliminary note) are presented. The chymotryptic peptides obtained from the reduced alkylated enzyme were purified and their amino acid sequences determined chiefly by the ‘Edman-dansylation’; procedure, and in two cases by partial acid or peptic hydrolyses. The tryptic peptides are alined into a single chain containing 129 amino acid residues, on the basis of overlapping peptides. Two labile glutamine residues easily converted into glutamic acid residues were characterized. Human milk lysozyme is compared with other human lysozymes (from normal and leuchaemic individuals) prepared by our group. The structure proposed is identical with the sequence of human leuchaemia lysozyme (from the urine of a patient with chronic monocytic leuchaemia) reported by Canfield. Human milk lysozyme is also near by related to several bird egg-white lysozymes (and bovine α-lactalbumin): Identical positions of Cys and Trp residues and of the residues essential for the catalytic activity or involved in some hydrogen bonds; several identical regions, especially in the β-sheet region; between 71 and 77 identical amino acid residues. It is suggested that by an insertion and a deletion in the sequence of human milk lysozyme, sequences homologous to those of bird lysozymes can be obtained.