Investigation of sorption capacity of dye-affinity sorbents in the process of purification of factor VIII coagulation

Background. Chromatographic methods, in particular affinity chromatography, are the most effective in obtaining highly purified preparations of plasma proteins. The active triazine and vinyl sulfone dyes are the special group of ligands for affinity chromatograph. We found that the dye-affinity sorbents bind non-target to factor VIII (FVIII) proteins in the previous stages of the study. The sorption capacity of the affinity sorbent is defined as the maximum amount of sample (protein) that can bind to the column under certain conditions. Determining the adsorption limit or limiting capacity of the sorbent makes it possible to identify the feasibility of its use to extract a certain type of protein. Objective. To study the sorption capacity of various dye-ligand affinity sorbents in the process of purification of FVIII. Materials and methods. We used next sorbents: Diasorb-Procion Blue HB, Diasorb-Procion Gelb M4R and Diasorb-Procion Blue MXR. The cryoprecipitate was initial material. The total protein concentration was determined by the Bradford method, the activity of factors VIII – one-stage clotting method. Results and discussion. Sorption of non-target proteins and FVIII activity were investigated after preparation of a number of dilutions of the initial solution of cryoprecipitate. Different concentrations of protein were applied per 1 cm3 of sorbent to select the optimal concentration and do not to oversaturate the column: I – 19.74±0.20 mg of protein/ml; II – 7.94±0.05 mg of protein/ml; III – 3.97±0.05 mg of protein/ml; IV – 1.96±0.04 mg of protein/ml. The maximum sorption capacity among the studied sorbents was 14.62±0.04 mg of protein / 1 cm3 for of sorbent Diasorb-Procion Blue HB. It was found that to achieve maximum purification of FVIII (highest specific activity), the optimal concentration of protein to 1 ml of sorbent should be in the range of 4-8 mg of protein / 1 cm3 of sorbent. The highest degree of purification for these sorbents was 19.65 times at an initial protein concentration of about 4 mg protein/ml (p≤0.01). Conclusions. The sorption capacity of sorbents was calculated. It was demonstrated that the maximum sorption capacity is approximately 15 (14.62±0.04) mg of protein / 1 cm3 of sorbent.

A New Solid-Phase Immunosorbent for Selective Binding of Desmoglein 3 Autoantibodies in Patients with Pemphigus Vulgaris

Autoantibodies, immunoglobulins G (IgG) againstthe desmosomal proteins desmogleins 1 and 3, play a significant role in the pathogenesis of pemphigus vulgaris. The basic therapy for pemfigus includes systemic corticosteroids, but their use should be as briefas possible because of the severe side effects. In cases of corticosteroid-resistant pemfigus, adjuvant therapy, in particularextracorporeal methods, is used. The most effective and safest extracorporeal therapy is immunosorbtion. Immunosorbtion is based on the removal of pemphigus antibodies from the blood using an affinity sorbent during a therapeutic apheresis procedure. Existing immunosorbents are nonselective and increase the risk of infection. We designed an immunosorbent based on an agarose matrix, Affi-Gel 15, and human recombinant desmoglein 3, as aligand, for a selective removal of autoantibodies from pemphigus patients sera. It was shown on a pemphigus experimental modelin vivo(neonatal Balb/c mouse model) andin vitrothat the immunosorbent can effectively remove desmoglein 3-associated autoantibodies. The experimental results demonstrate that the solid-phase matrix immunosorbent Affi-Gel 15Dsg3 is a promising product for the development of pemphigus therapy.

The effect of a neuroprotective dose of isatin or deprenyl to mice on the profile of brain isatin-binding proteins

Isatin (indol-2,3-dione), an endogenous biofactor found in the brain, peripheral tissues and biological body fluids of humans and animals, exhibits a wide range of biological and pharmacological activities. They are realized via interaction with numerous isatin-binding proteins. Some of these proteins identified during proteomic profiling of the brain are involved in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl (selegiline), a pharmacological agent used for treatment of Parkinson's disease. In this study, we have investigated the effect of a single dose administration of isatin (100 mg/kg) and deprenyl (10 mg/kg) to mice on the profile of the brain isatin-binding proteins. Comparative proteomic analysis of brain isatin-binding proteins of mice treated with isatin or deprenyl resulted in identification of a representative group of proteins (n=200) sensitive to the administration of these substances. The change in the profile of isatin-binding proteins may be obviously attributed to accumulation of isatin and deprenyl in the brain and their interaction with target proteins; this prevents protein binding to the affinity sorbent. Thus identified brain isatin-binding proteins of the control animals obviously represent specific targets that interact directly with isatin (and also with deprenyl) in vivo. Isatin or deprenyl administered to animals interact with these proteins and thus inhibit their binding to the affinity sorbent (immobilized isatin analogue).

The effect of deprenyl and isatin administration to mice on the proteomic profile of liver isatin-binding proteins

Isatin (indol-2,3-dione) is an endogenous indole found in the brain, peripheral tissues and biological body fluids of humans and animals. Its wide spectrum of biological activity is realized via interaction with numerous isatin-binding proteins; these include proteins playing an important role in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl, a pharmacological agent used for treatment of Parkinson's disease. In this study, the effects of the course of deprenyl (1 mg/kg) and isatin (20 mg/kg) administration for 21 days on the profile of the isatin-binding proteins of the liver of mice have been investigated. Proteomic profiling of liver isatin-binding proteins of control mice by means of 5-aminocaproylisatin as an affinity ligand resulted in identification of 105 proteins. Treatment of animals with a low dose of isatin slightly decreased (up to 91), while injections of deprenyl slightly increased (up to 120) the total number of isatin-binding proteins. 75 proteins were common for all three groups; they represented from 62.5% (in deprenyl treated mice) and 71% (in control mice), to 82% (isatin treated mice) of the total number of identified liver isatin-binding proteins. Proteomic analysis of the isatin-binding proteins of mice treated with isatin (20 mg/kg) or deprenyl (1 mg/kg) for 21 days revealed a representative group of proteins (n=30) that were sensitive to the administration of these substances. Taking into account the previously obtained results, it is reasonable to suggest that the change in the profile of isatin-binding proteins may be attributed to accumulation of isatin and deprenyl in the liver and interaction with target proteins prevents their subsequent binding to the affinity sorbent. In this context, the identified isatin-binding liver proteins of control animals that do not bind to the affinity sorbent (immobilized isatin analogue) after treatment of animals with either deprenyl or isatin appear to be specific targets directly interacting with isatin in vivo.