Use of ion-exchange and hydrophobic-interaction chromatography for the rapid purification of lysozyme—estrone glucuronide conjugates

Smales, C. Mark; Cooke, Delwyn G.; Blackwell, Leonard F.

doi:10.1016/0378-4347(94)00381-5

Cited by 9 publications

(16 citation statements)

References 18 publications

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“…Commercial hen egg-white lysozyme (Grade 1, three times recrystallized and lyophilized; Sigma Co., Poole, Dorset, UK) was further purified before use to remove a small contaminant peak, as previously described (Smales et al, 1994). The trypsin used in digestion experiments was of sequencing-grade quality and was also from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Protein modification during antiviral heat bioprocessing

Smales

Pepper

James

2000

Biotechnol. Bioeng.

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Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg‐white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet‐ and dry‐heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography–electrospray ionization–mass spectroscopy (LC‐ESI‐MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet‐heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys97, by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 177–188, 2000.

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Section: Methodsmentioning

confidence: 99%

Protein modification during antiviral heat bioprocessing

Smales

Pepper

James

2000

Biotechnol. Bioeng.

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“…[H] was achieved by both the mixed anhydride procedure of Erlanger et al (20) and the N-hydroxysuccinimide/dicyclohexylcarbodiimide coupling method (21) at a 1.6:1 molar ratio of steroid glucuronide to lysozyme as described previously (19). The mixed anhydride reagent was prepared from E1G[H] (7.2 mg, 16.1 µmol) in dimethylformamide (116 µL) in a 0.5 mL glass reaction vial.…”

Section: Preparation Of Estrone Glucuronide (E1g[h])-lysozyme Conjuga...mentioning

confidence: 99%

“…Purification of Estrone Glucuronide-Lysozyme Conjugates. The success of the conjugation experiments and purity of the derivatives was determined by analytical chromatography on a Mono-S cation-exchange column in 7 M urea buffers (19). Estrone glucuronide-lysozyme conjugates were purified from the conjugation reaction mixture by cation-exchange chromatography, also in 7 M urea buffers, on an S-Sepharose (fast flow) column (50 × 1.6 cm i.d.)…”

Section: Preparation Of Estrone Glucuronide (E1g[h])-lysozyme Conjuga...mentioning

confidence: 99%

“…The following reagents were obtained from the sources indicated: 17-oxoestra-1,3,5-3-β-yl-D-glucopyranosiduronic acid (estrone glucuronide or E1G[H]) was synthesized essentially according to Conrow and Bernstein (18) as described previously (19). Commercial hen egg white lysozyme (Grade 1, three times recrystallized and lyophilized, Sigma, St. Louis, MO) was further purified before use as described previously (19). Antisera were raised in sheep against thyroglobulin-estrone glucuronide conjugates.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Lysozyme-Estrone Glucuronide Conjugates. The Effect of the Coupling Reagent on the Substitution Level and Sites of Acylation

1999

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Estrone glucuronide conjugates of hen egg white lysozyme were prepared by the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugates, but the active ester procedure gave a more diverse range of products, making it less suitable for preparing conjugates for homogeneous enzyme immunoassay. Conjugation of lysozyme with estrone glucuronide by the mixed anhydride procedure gave one major derivative exclusively acylated at lysine residue 33 whereas conjugation by the active ester method gave six derivatives which were acylated at one or more of lysine residues 33, 97, and 116. None of the lysine residues 1, 13, and 96, or the N-terminal alpha-amino group, were acylated in any of the conjugates isolated. The correlation of the conjugate structures with the protein environments of the amino groups in the crystal structure of lysozyme suggested that the sites of acylation were determined not only by the chemical nature of the acylating reagent but also by the surface accessibility and nucleophilicity of the individual lysine residues.

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“…Also, the structural similarity of T4L with HEWL is relatively low and it would, therefore, also require much re-working of the reported conjugation and purification procedures. [4,5,7,23] The small increase in lytic rate obtained with TEWL ( Table 2) was insufficient to justify the work required to prepare and test TEWL-E1G conjugates for use as signal generators, even though it has high structural similarity with HEWL.…”

Section: Discussionmentioning

confidence: 94%

Clearing of Suspensions ofMicrococcus lysodeikticusCatalysed by Lysozymes from Hen, Goose, and Turkey Egg Whites, Human Milk, and Phage T4. Assessment of Potential as Signal Generators for Homogeneous Enzyme Immunoassays for Urinary Steroids

Cooke

Blackwell

2007

Journal of Immunoassay and Immunochemistry

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Lysozymes (3.2.1.17) from goose (Anser anser) egg white, turkey (Melagris gallopavo) egg white, phage T4 and human milk were compared with hen egg white lysozyme in their ability to clear a suspension of Micrococcus lysodeikticus. All of the lysozymes, except hen egg white lysozyme, catalysed the clearing of the Micrococcus lysodeikticus suspension in a biphasic fashion. Compared to hen egg white lysozyme, the total absorbance or transmission change over 5 and 20 minutes was less in all cases, except for human lysozyme. Human lysozyme was, therefore, a potential alternative, more rapid signal generator for the measurement of urinary estrone glucuronide excretion rates because of its structural similarity to hen egg white lysozyme. The apparent K(M) values for hen egg white lysozyme increased with the enzyme concentration.

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Use of ion-exchange and hydrophobic-interaction chromatography for the rapid purification of lysozyme—estrone glucuronide conjugates

Cited by 9 publications

References 18 publications

Protein modification during antiviral heat bioprocessing

Protein modification during antiviral heat bioprocessing

Characterization of Lysozyme-Estrone Glucuronide Conjugates. The Effect of the Coupling Reagent on the Substitution Level and Sites of Acylation

Clearing of Suspensions ofMicrococcus lysodeikticusCatalysed by Lysozymes from Hen, Goose, and Turkey Egg Whites, Human Milk, and Phage T4. Assessment of Potential as Signal Generators for Homogeneous Enzyme Immunoassays for Urinary Steroids

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