Mapping of Recombinant Hemoglobin Using Immobilized Trypsin Cartridges

We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the ␤-and di-␣-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNA Leu , similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

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“…Tryptic mapping was performed using Porozyme trypsin cartridges (25)(26). Separation was performed using a Zorbax C18 column.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli

Apostoł¹,

Levine²,

Lippincott³

et al. 1997

Journal of Biological Chemistry

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100

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“…Tryptic Mapping-Tryptic mapping was performed as described by Lippincott et al (34). The percent change from rHb1.1 referred to in Table II (35).…”

Section: Hemoglobin Characterizationmentioning

confidence: 99%

“…Reverse Phase HPLC-Samples were prepared by precipitation with ice-cold acid/acetone as described by Witkowska et al (36) and Lippincott et al (34). Pellets were solubilized in 0.1% trifluoroacetic acid, 20% acetonitrile at a final concentration of 1 mg/ml.…”

Section: Hemoglobin Characterizationmentioning

confidence: 99%

“…LC-MS-Mass spectrometry was performed as described by Lippincott et al (34) using a Finnigan Mat LCQ with an HP1090 HPLC on the front end to run reverse phase separations prior to analysis.…”

Section: Hemoglobin Characterizationmentioning

confidence: 99%

“…The notation of Lippincott et al (34) is used here to denote tryptic fragments. The tryptic profiles of the mono-and poly-glxrHb fractions were similar but demonstrated significant differences when compared with unmodified rHb1.1 (Fig.…”

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confidence: 99%

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Glutaraldehyde Modification of Recombinant Human Hemoglobin Alters Its Hemodynamic Properties

Doyle

Apostoł

Kerwin

1999

Journal of Biological Chemistry

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Many cell-free hemoglobin solutions designed as oxygen-carrying therapeutics produce a hypertensive effect in animals. The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by glutaraldehyde would attenuate the hypertensive response. Two products of the cross-linking reaction were isolated, a glutaraldehyde-derivatized monomer (monoglxrHb) and a glutaraldehyde cross-linked polymer (poly-glxrHb), and evaluated for their effects on systemic hemodynamics in conscious rats. Administration of rHb1.1 caused a mean arterial pressure elevation of approximately 20 mm Hg and an increase in total peripheral resistance of approximately 30%. Administration of mono-glxrHb induced changes in mean arterial pressure and vascular resistance that were significantly diminished relative to those observed with rHb1.1. PolyglxrHb elicited a mean arterial pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same as the response to mono-glxrHb. These results suggest that rHb peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and that the effect of glutaraldehyde polymerization is likely due to surface modification and/or intramolecular cross-linking, rather than an increase in molecular size.As reviewed elsewhere (1-3), the search for a safe and efficacious oxygen-delivering therapeutic has been ongoing for many years. Limitations to the use of hemoglobin solutions as therapeutics have included renal toxicities due to the presence of stromal elements (4, 5), dissociation into ␣␤ dimers (6), high oxygen affinities (7), and short intravascular retention times (1). Multiple approaches have been taken to modify hemoglobins to address these limitations (1, 3), including chemical cross-linking of purified human or bovine hemoglobin to prevent dissociation and chemical modification to increase the P 50 to a physiologically suitable level (approximately 25-35 mm Hg, (7)). Recombinant technology was employed to genetically cross-link the ␣-globins to form a di-␣-globin and thereby prevent dissociation. In addition, the P 50 was increased by substitution of Lys for Asn at the ␤108 position. The resultant hemoglobin molecule, rHb1

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Proteolytic Mapping

Nadler¹

2000

Encyclopedia of Analytical Chemistry

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Proteolytic mapping is a technique used to verify the primary structure of a protein. It involves the cleavage of the protein at specific amino acids to generate a unique set of peptides which is then separated and analyzed. The chromatogram generated during the separation is known as the proteolytic map of the protein, which serves as a unique “fingerprint” for that protein. A proteolytic map can be used to verify the identity of a protein and to detect mutations in a protein. This technique is faster than two‐dimensional (2D) nuclear magnetic resonance (NMR), X‐ray crystallography, or complete amino acid sequencing for verifying the primary structure of a protein. However, it does not allow determination of the sequence of a protein de novo as can be accomplished with sequencing, nor does it yield the higher order structural information that may be obtained with 2D NMR or X‐ray crystallography. The process of proteolytic mapping involves sample preparation, enzyme digestion, and map development. Each of these steps should be optimized for each new protein to obtain a good map; alternatively, generic protocols can be used to produce satisfactory results for some proteins. A typical protocol requires about a day to complete owing to the time required to digest a protein with an enzyme. However, more recent automated techniques are capable of completing the process in a few hours because they employ a highly concentrated immobilized enzyme which speeds digestion. Proteolytic maps are used in both quality control (QC) and research environments, with the most recent application in the field of proteomics.

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Mapping of Recombinant Hemoglobin Using Immobilized Trypsin Cartridges

Cited by 37 publications

References 17 publications

Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli

Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli

Glutaraldehyde Modification of Recombinant Human Hemoglobin Alters Its Hemodynamic Properties

Proteolytic Mapping

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