Sickle Hemoglobin Polymer Melting in High Concentration Phosphate Buffer

Louderback, Joseph G.; Ballas, Samir K.; Kim‐Shapiro, Daniel B.

doi:10.1016/s0006-3495(99)77377-7

Cited by 10 publications

(4 citation statements)

References 31 publications

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“…HbS polymerization and gelation, which occur when HbS is in deoxy-state in the venous circulation, are considered the primary pathogenic event of sickle cell anemia (10)(11)(12)(13)(14)(15). Hence, the thermodynamics of HbS solutions (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and the formation of the polymers and of other condensed phases (9,(26)(27)(28)(29)(30)(31) are studied as part of the fundamental mechanisms of the disease. These investigations have revealed that over periods of several days, HbS solutions form crystals (32)(33)(34).…”

Section: Introductionmentioning

confidence: 99%

Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin

Pan

Galkin

Filobelo

et al. 2007

Biophysical Journal

112

208

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Sickle cell hemoglobin (HbS) is a mutant, whose polymerization while in deoxy state in the venous circulation underlies the debilitating sickle cell anemia. It has been suggested that the nucleation of the HbS polymers occurs within clusters of dense liquid, existing in HbS solutions. We use dynamic light scattering with solutions of deoxy-HbS, and, for comparison, of oxy-HbS and oxy-normal adult hemoglobin, HbA. We show that solutions of all three Hb variants contain clusters of dense liquid, several hundred nanometers in size, which are metastable with respect to the Hb solutions. The clusters form within a few seconds after solution preparation and their sizes and numbers remain relatively steady for up to 3 h. The lower bound of the cluster lifetime is 15 ms. The clusters exist in broad temperature and Hb concentration ranges, and occupy 10(-5)-10(-2) of the solution volume. The results on the cluster properties can serve as test data for a potential future microscopic theory of cluster stability and kinetics. More importantly, if the clusters are a part of the nucleation mechanism of HbS polymers, the rate of HbS polymerization can be controlled by varying the cluster properties.

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Section: Introductionmentioning

confidence: 99%

Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin

Pan

Galkin

Filobelo

et al. 2007

Biophysical Journal

112

208

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“…It was suspected that the structural changes of HbS due to polymerization under hypoxic conditions might be reflected in the form of a variation in absorption [17]. Therefore, the absorption spectrum of the HbS protein was recorded and compared under hypoxia and normoxia conditions.…”

Section: Characterization Of Polymerization Of Hemoglobin S Under Hyp...mentioning

confidence: 99%

Novel Use of Hypoxia-Inducible Polymerizable Protein to Augment Chemotherapy for Pancreatic Cancer

et al. 2022

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and is the fourth leading cause of cancer-related deaths in the United States. Unfortunately, 80–85% of patients are diagnosed with unresectable, advanced stage tumors. These tumors are incurable and result in a median survival less than approximately six months and an overall 5-year survival rate of less than 7%. Whilst chemotherapy is a critical treatment, cure is not possible without surgical resection. The poor clinical outcomes in PDAC can be partially attributed to its dense desmoplastic stroma, taking up roughly 80% of the tumor mass. The stroma surrounding the tumor disrupts the normal architecture of pancreatic tissue leading to poor vascularization, high intratumoral pressure along with hypoxia and an acidic tumor microenvironment. This complicated microenvironment presents a significant challenge for drug delivery. The current manuscript discusses a novel approach to overcome many of these various obstacles. A complex of gemcitabine (GEM) and hemoglobin S (HbS) was formulated, which self-polymerizes under hypoxic and acidic conditions. When polymerized, HbS has the potential to break the tumor stroma, decrease intratumoral pressure, and therefore improve the treatment efficacy of standard therapy. Intratumoral injection of HbS with a fluorescent small molecule surrogate for GEM into a pancreatic tumor xenograft resulted in improved dissemination of the small molecule throughout the pancreatic tumor. The self-polymerization of HbS + GEM was significantly more effective than either agent individually at decreasing tumor size in an in vivo PDAC mouse model. These findings would suggest a clinical benefit from delivering the complex of GEM and HbS via direct injection by endoscopic ultrasound (EUS). With such a treatment option, patients with locally advanced disease would have the potential to become surgical candidates, offering them a chance for cure.

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“…The assay is fast and economic screening technique that does not use whole sickle blood, rather commercially available purified sickle hemoglobin provided by Sigma-Aldrich. Hobs polymerization was achieved at a very low concentration of 50.0 µM by conducting the assay in high phosphate buffer (1.5M) medium [19]. Using as little as 250 µl of 40 mg/ml HBS solution; a given test compound can be evaluated at 5 different concentrations; each in quadruple, in a period of 20 minutes.…”

Section: Current Assaymentioning

confidence: 99%

High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin Polymerization

Mehanna¹

2017

Med chem

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The current manuscript describes a high throughput assay designed to identify organic compounds with potential inhibitory effects on sickle hemoglobin polymerization. The assay is fast, economic and reproducible. In just 20-minutes, a test compound can be screened for anti-polymerization activity at five different concentrations; each in quadruple; using as little as 10 mg of purified sickle hemoglobin. The assay was conducted in high phosphate buffer concentration (1.5M), a concentration that allows sickle hemoglobin to polymerize at a very low concentration of 50.0 µM. The new assay was validated by evaluating the inhibitory effects of the amino acid phenylalanine, a standard control used in all gelation assays, and hydroxyl urea, the only FDA approved drug to treat sickle cell anemia. Phenylalanine showed a reproducible and concentration-dependent delay of sickle hemoglobin polymerization at a concentration range of 25-75mM. Hydroxyurea; although its action is thought to be through promoting fetal hemoglobin formation, was found to have direct inhibitory effects on sickle hemoglobin polymerization but at a high concentration range of 64-500mM. The assay was applied for random screening of several organic compounds and 2-thio-salicylic acid was identified to be a powerful inhibitor for polymerization at much lower concentration range of 0.1-1.6 mM.

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Sickle Hemoglobin Polymer Melting in High Concentration Phosphate Buffer

Cited by 10 publications

References 31 publications

Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin

Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin

Novel Use of Hypoxia-Inducible Polymerizable Protein to Augment Chemotherapy for Pancreatic Cancer

High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin Polymerization

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