Separation of hemoglobins and hemoglobin chains by high-performance liquid chromatography

Huisman, T. H. J.

doi:10.1016/0378-4347(87)80012-9

Cited by 86 publications

(18 citation statements)

References 34 publications

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“…We carried out HPLC analysis 20 of cell lysates from A06, A07, A09, and healthy individuals ( Figure 7). Normal cells contain 1% HbF and A07 has 4% HbF, whereas A09 and A06 have 31% and 63% HbF, respectively.…”

Section: ␥ Mrna Level Is the Major Determinant Of Hbf Expressionmentioning

confidence: 99%

The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression

Chakalova

Osborne

Dai

et al. 2005

Blood

112

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Section: ␥ Mrna Level Is the Major Determinant Of Hbf Expressionmentioning

confidence: 99%

The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression

Chakalova

Osborne

Dai

et al. 2005

Blood

112

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“…They were not resolved by high performance anion exchange chromatography, although this technique can resolve a variety of soluble (20,21) and membrane bound (9, 20) Cyt P-450 in extracts from other organisms. ARP-1 and ARP-2 can be separated by reverse phase HPLC, which is capable of resolving smaller proteins differing by as little as a single amino acid (14,25).…”

Section: Substrate Binding and Enzymatic Activitymentioning

confidence: 99%

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

O’Keefe

Leto²

1989

Plant Physiol.

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The microsomal fraction from the mesocarp of avocado (Persea amerkana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest pattems, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarites with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.Cytochrome P450 dependent monooxygenases are widespread in nature. They are involved in a variety of catabolic and biosynthetic pathways and in the metabolism of drugs and xenobiotics. In plants, Cyt P-450 has been identified, for example, in the trans-cinnamic acid and kaurene hydroxylases (10,11). Others are suspected participants in a number of reactions associated with xenobiotic metabolism (6, 8) (for reviews see Refs. 4 and 20). Within these monooxygenase systems, Cyt P-450 serves as a terminal oxidase, responsible for substrate recognition, binding, and oxygen redox chemistry (5, 29).The mechanistic and structural details of Cyt P450 dependent monooxygenases have been developed largely from studies of the bacterial (Pseudomonas putida) camphor hydroxylase system (23,29). Because of their importance in xenobiotic and drug metabolism, these enzymes have also been thoroughly studied in mammalian liver (3,5,31). In contrast, Cyt P-450 of higher plant origin has not been the subject of extensive biochemical characterization, primarily because of the low content in many plant tissues, difficulties with identification in Chl containing tissues, and supposed lability in homogenized plant extracts. ' (6,10,12,13,19,20). Cyt P-450 has been purified from tulip (Tulipa gesneriana) bulbs (12), and from tubers of Jerusalem artichoke (Helianthus tuberosus) (10). Additionally, an NADPH:Cyt P450 reductase has been purified from Jerusalem artichoke (2), and the related Cyt b5 and NADH:Cyt b5 reductase have been purified from Pisum sativum (16). Unfortunately, the tulip bulb Cyt P450 has not been associated with any enzymic activity, and polyclonal antibodies raised against it were not cross reactive with proteins from other species (1 1). The Jerusalem artichoke preparation can be reconstituted into its trans-cinnamate hydroxylase activity, but it has a low heme specific content and is not homogeneous (10). No primary sequence information has been reported for either o...

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“…Blood samples were diluted (1:250) in H 2 O Chromasolv for HPLC (Sigma-Aldrich, St. Louis, Mo., USA) and, after spinning for 1 min in a microcentrifuge, the supernatant was injected into the HPLC system [16, 20]. Cells were washed with PBS and the obtained pellet was lysed in lysis buffer (SDS 0.01%).…”

Section: Methodsmentioning

confidence: 99%

“…After spinning for 1 min, the supernatant was collected and injected. Hb proteins present in blood samples and in the hemolysates were separated by cation-exchange HPLC [20], using a Beckman Coulter instrument System Gold 126 Solvent Module-166 Detector. Hb were separated using a Syncropak CCM 103/25 (250 × 4.6 mm) column, samples were eluted in a solvent gradient utilizing aqueous sodium acetate-BisTris-KCN buffers and detection was performed at 415 nm.…”

Section: Methodsmentioning

confidence: 99%

A Novel Frameshift Mutation (+A) at Codon 18 of the β-Globin Gene Associated with High Persistence of Fetal Hemoglobin Phenotype and δβ-Thalassemia

Feriotto

Salvatori²,

Finotti³

et al. 2008

Acta Haematol

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We report in this paper a novel thalassemia mutation (insertion of a single A nucleotide within the exon 1, at codon 18, of the β-globin gene) associated with a deletion of the δβ-globin gene region, in a patient exhibiting high persistence of fetal hemoglobin. The novel mutation causes a frameshift with the generation of a UGA stop codon. Analysis of the parent’s DNA demonstrates that the A insertion and frameshift mutation are inherited from the father, while the δβ-globin gene deletion is inherited from the mother. Gene dosage analysis and deletion-specific PCR demonstrate that the deletion is the (δβ)⁰ Sicilian deletion, involving a 13.4-kb δβ-globin gene region.

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Separation of hemoglobins and hemoglobin chains by high-performance liquid chromatography

Cited by 86 publications

References 34 publications

The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression

The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

A Novel Frameshift Mutation (+A) at Codon 18 of the β-Globin Gene Associated with High Persistence of Fetal Hemoglobin Phenotype and δβ-Thalassemia

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