Chapter 24 Protein Chromatography on Hydroxyapatite Columns

Cummings, L. J.; Snyder, Mark A.; Brisack, Kimberly

doi:10.1016/s0076-6879(09)63024-x

Cited by 89 publications

(53 citation statements)

References 39 publications

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“…1 are strikingly similar to images previously obtained by immunohistochemical staining of proteins earlier identified as constituents of sub-RPE deposits, such as amyloid beta (15,16), CFH (31), and vitronectin (32). HAP is well known to bind proteins, and for this reason, it is widely used as a stationary phase for chromatography (33). Consequently, we tested the hypothesis that the HAP spherules promote the growth of the protein-rich sub-RPE deposits by binding proteins present in the sub-RPE space.…”

Section: Significancesupporting

confidence: 84%

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

Thompson

Reffatto

Bundy³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

113

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Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5–20 μm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch’s membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch’s membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits.

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Section: Significancesupporting

confidence: 84%

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

Thompson

Reffatto

Bundy³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Since HA columns are normally operated at pH 6.8 after extensive washing with a phosphate buffer, the surface of the column can be regarded as negative because of a partial neutralization of the positive calcium loci by phosphate ions. 10 Lysozyme is a basic protein (pI 5 11.0). In the adsorption medium (50 mM at pH 7.0 in phosphate buffer), the protein surface will be positively charged, and the adsorbent surface will be negatively charged.…”

Section: Articlementioning

confidence: 99%

“…[7][8][9] Porous type I CHT adsorbed larger amounts of lysozyme compared with type II CHT because of its lower surface area. 10 Among these studies, only a limited number of systems use magnetic HA for protein separation in a magnetically stabilized fluidized bed (MSFB) system. One of these studies used BcMag HA modified magnetic beads in a batch mode.…”

Section: Introductionmentioning

confidence: 99%

Separation of lysozyme with magnetically stabilized spherical hydroxyapatite microcomposites in a continuous flow system

Akkaya

2013

J of Applied Polymer Sci

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The aim of this study was to produce a spherical and magnetic hydroxyapatite (HA) microcomposite to use as an adsorbent for the separation of lysozyme in a magnetically stabilized fluidized bed and to separate lysozyme from a protein mixture composed of human immunoglobulin G, human serum albumin, and lysozyme. For this purpose, spherical and magnetic HA microcomposites (50-100 lm) were synthesized by suspension polymerization and characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, electron spin resonance, vibrating-sample magnetometry, Brunauer-Emmett-Teller analysis, and a swelling test. The specific surface areas of the pure HA and magnetic HA microcomposite were determined to be 72.25 and 151.53 m 2 /g, respectively. The swelling ratio of the spherical HA microcomposites was 150%. Adsorption experiments were conducted under conditions that differed in terms of pH, temperature, ionic strength, flow velocity, and magnetic field. According to the findings of the adsorption kinetic studies, the adsorption process was appropriate to pseudo-first-order kinetics. The separation of the lysozyme from the protein mixture was achieved by means of a 50 mM pH 7.0 phosphate buffer.

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“…Although hydroxyapatite has very well-known affinity for proteins -as proved from the wide use of chromatographic HA columns for protein separation- [8][9][10] , the selectivity in protein adsorption is greatly determined by the surface charge that HA develops in the physiological environment.…”

Section: Introductionmentioning

confidence: 99%

Impact of Porosity and Electrolyte Composition on the Surface Charge of Hydroxyapatite Biomaterials

Español

Mestres

Luxbacher

et al. 2016

ACS Appl. Mater. Interfaces

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The success or failure of a material when implanted in the body is greatly determined by the surface properties of the material and the host tissue reactions. The very first event that takes place after implantation is the interaction of soluble ions, molecules and proteins from the biological environment with the material surface leading to the formation of an adsorbed protein layer that will later influence cell attachment. In this context, the particular topography and surface charge of a material become critical as they influence the nature of the proteins that will adsorb. However, very limited information is available on the surface charge of porous substrates. Only until very recently the determination of the zeta potential on porous membranes has been accurately determined. The goal of this work was to implement the previous findings for the determination of the zeta potential of a series of porous hydroxyapatite (HA) substrates and to assess how porosity affects the measurements. In addition, studies using various electrolytes were also performed to prove how the specific affinity of certain ions for HA can further impact surface charge. The results showed that all materials exhibited very similar external surface charge (~ -23 mV), consistent with their almost identical topographies.However, the presence of interconnected pores underneath the sample surface resulted in an additional internal zeta potential that varied with the porosity content. Measurements with different electrolytes confirmed the selectivity of divalent ions for HA underlying the importance of testing biomaterials using relevant electrolytes.3

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Chapter 24 Protein Chromatography on Hydroxyapatite Columns

Cited by 89 publications

References 39 publications

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

Separation of lysozyme with magnetically stabilized spherical hydroxyapatite microcomposites in a continuous flow system

Impact of Porosity and Electrolyte Composition on the Surface Charge of Hydroxyapatite Biomaterials

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