Characterization of hemoglobin immobilized on gamma-zirconium phosphate

The fact that different γ-zirconium phosphate (γ-ZrP) preintercalation method induced varied degree and type of conformational change of the adsorption protein was confirmed by characterization techniques including circular dichroism (CD), fourier transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD) analysis. The results indicated that the association of hemoglobin with γ-ZrP preintercalated using butylamine was correlated with conformational change in the secondary structure of the protein. γ-ZrP which was preintercalated with tetra (n-butylammonium) hydroxide caused the conformational change of Hemoglobin in both the secondary structure and the tertiary structure. X-ray powder diffraction analysis was used to analyze the crystalline structure of the nanocomposites prepared by relamination. The adsorption isotherms of Hemoglobin on different matrices were set up and fitted with Langmuir and Freundlich equations.     

Immobilized hemoglobin in the purification of hemoglobin-based oxygen carriers.

Chemically modified hemoglobins can be used as oxygen carriers in cell-free fluids provided that they have a low oxygen affinity and are stable towards dissociation into subunits. The latter species are undesirable because they are filtered rapidly through the kidneys, have renal toxicity and are characterized by a high oxygen affinity. A most important step in the preparation of hemoglobin-based oxygen carriers is therefore their purification from any dissociable material. Hemoglobin immobilized as alpha beta dimers on Sepharose lends itself naturally to this purpose as it is able to interact in a specific and reversible way with soluble alpha beta dimers. Hemoglobin affinity columns are very effective in the purification of cross-linked and pseudo-cross-linked human and bovine hemoglobin. The applicability of the technique is enhanced by the ease with which alpha beta dimers from different species cross-interact to yield hybrid alpha 2 beta 2 tetramers. It is shown that hemoglobin affinity columns may provide analytical information on the cross-linking reaction itself.     

Electrochemical studies of rutin interacting with hemoglobin and determination of hemoglobin

Electrochemical investigation of the interaction of rutin (Rt) with hemoglobin (Hb) at a glassy carbon electrode is reported for the first time. With the addition of hemoglobin in a rutin solution, both the reduction and oxidation currents decrease with few changes in the peak potentials. The reaction of rutin with hemoglobin forms a nonelectroactive supramolecular complex Hb-Rt. The equilibrium constant for complex is calculated to be 3.4x10(6). A satisfactory result has been obtained for the determination of hemoglobin in clinical blood samples.     

An electrochemical biosensor for nitric oxide based on silver nanoparticles and hemoglobin.

A nitric oxide (NO) biosensor based on silver nanoparticles was fabricated with high sensitivity and selectivity as well as stability. Silver nanoparticles could preserve the microstructures of hemoglobin, but the electrochemical reactivity of the protein and its detection sensitivity toward NO could be greatly enhanced. Accordingly, a NO biosensor was developed. The linear concentration range was from 1.0 x 10(-6) to 5.0 x 10(-5) M. Its detection limit was 3.0 x 10(-7) M with a sensitivity of 0.0424 microA microM(-1) NO. The possible co-existing compounds would not interfere with the detection.     

Electrochemical investigation of the chloride effect on hemoglobin

Direct electron transfer between hemoglobin and gold electrode is achieved at both a bare and a 4, 4'-bipyridine-modified gold electrode in the presence of chloride ions. The addition of chloride to hemoglobin solution also increases the reversibility of the direct electrochemistry and shifts the formal potential of hemoglobin to the negative direction. While the existence of chloride does not significantly change the tertiary structure of the protein, it might induce a slight variation of the structure, which is beneficial to the electrochemical response. It is suggested that the chloride binding to hemoglobin is a combination of specific and unspecific bindings.     

Electrochemical studies of 9,10-anthraquinone interacting with hemoglobin and determination of hemoglobin

Electrochemical investigation of the interaction of 9,10-anthraquinone (AQ) with hemoglobin (HB) on a mercury electrode is reported for the first time. On addition of hemoglobin to an anthraquinone solution, both the reduction and oxidation currents decrease, with increasing peak separation. In the presence of hemoglobin, no new peaks appear, but the electrochemical parameters (standard rate constantk _s and diffusion coefficientD) change significantly. Reaction of anthraquinone with hemoglobin forms an electrochemically active complex HB-AQ. The equilibrium constant for this complex is calculated to be 3.27 × 10⁵ l/mol. A satisfactory result has been obtained for the determination of hemoglobin in clinical blood samples.     

Biologically active polymer systems based on hemoglobin

Development of the scientific principles involved in the creation of soluble macromolecular modifications of the native protein, hemoglobin, is surveyed. The main goal of the research is practical application of the results to develop blood substitutes with oxygen-transport function for treatment of massive blood loss. Models of red blood cells, which represent corpuscular polymer systems (microcapsules, liposomes, microdispersions) are considered.     

Adsorption properties of hemoglobin

The isotherms of hemoglobin adsorption on adsorbents of different nature and porous structure are obtained at different adsorption times. The maximum adsorption values are determined, and the adsorption’s relationship with the adsorbent pore size is established. The possibility of using hemoglobin as a test protein to determine the adsorbent surface area available for protein monolayer formation is demonstrated.     

Simple method for the isolation and purification of hemoglobin components.

A simple method for the isolation and purification of hemoglobin components from starch gel by electrophoresis is described. An inverted bottle with a cut off bottom is used. Pieces of gel containing the hemoglobin are embedded in potato starch, wetted with buffer, and placed in the neck of the inverted bottle, the mouth of which has been closed with filter paper and tape. Connection with the negative tank buffer is achieved by inserting a layer of starch gel between the potato starch and the negative tank buffer. By electrophoresis, the hemoglobin migrates and accumulates in a dialysing tube tied around the mouth of the bottle and hanging into the positive tank buffer.     

Effect of different proteases on bitterness of hemoglobin hydrolysates

Hemoglobin was hydrolyzed by several enzymes (Proctase, Alcalase, Neutrase, papain). Hydrolysates were analyzed (degree of hydrolysis, gel permeation on Superose 12 column, tasting) and fractionated by ultrafiltration and 2-butanol extraction. The bitter peptides were isolated and identified. The results were compared with those already obtained with peptic hemoglobin hydrolysates. All the findings were confirmed. Ultrafiltration concentrated bitter compounds in the fraction corresponding to 500–5000 Da, and these compounds were selectively extracted by 2-butanol. All the bitter peptides belonged to the same fragment of the Β-chain of bovine hemoglobin. Finally, the use of a Superose 12 Chromatographic column for easy detection of bitter hydrolysates without sensory analysis could be generalized for hemoglobin hydrolysates.     

Vibronic coupling effect on the paramagnetism of deoxygenated hemoglobin

A square pyramidal iron(II) complex coupled with a β₁ vibrational mode is considered as a model for deoxyhemoglobin. The calculations performed on this model show that the introduction of the vibronic coupling may allow a strong reduction of the rhombic splitting and account for the different magnetic moment in phosphate-free and phosphate-bound deoxyhemoglobin.     

Capillary electrophoretic determination of cathepsin D activity using Oregon Green-labeled hemoglobin.

Cathepsin D is an aspartyl protease of lysosomal origin and functions in a variety of roles including protein turnover, catabolism of peptide hormones, antigen processing and presentation, and neoplastic disease. In breast cancer, the level of cathepsin D has been linked to metastasis and prognosis for survivability. Many of these studies concerning the role of cathepsin D in cancer have used immunological detection methods to determine the level of enzyme. These indirect methods to assess the cathepsin D level may not reflect enzyme activity accurately. The significance of cathepsin D to physiological and pathophysiological processes suggests that rapid and sensitive methods for determining cathepsin D activity would contribute to a more complete assessment of this enzyme in its various roles. This work describes a procedure to determine cathepsin D activity based on hydrolysis of fluorescently labeled hemoglobin and employs capillary electrophoresis to separate and measure the products of reaction. A single major cleavage product, representing the first 32 residues of the hemoglobin alpha-chain, appeared after a very short incubation time (less than 10 min) and was used to determine activity. The procedure described here requires very small sample volumes, has a low detection limit (approximately 10(-9) M) and thus represents an additional approach to determine cathepsin D activity in biological samples.