Simple two-step semi-preparative isolation and purification of transferrin from human serum

Human transferrin (Tf) is a bilobal 76 kDa iron-binding glycoprotein present in human serum. Each lobe has the ability to bind one ferric ion (Fe3+) and a single synergistic bicarbonate anion. The main role of Tf is to transport Fe3+ ions through the circulation to cells, via interaction with transferrin receptor (TFR) on the cell surface. Previously described methods for Tf isolation and purification are either very time-consuming or provide Tf of lower final purity. Here we describe a fast and simple FPLC method for the isolation and purification of Tf from human serum. Serum samples were prepared by precipitation, while protein purification was performed on FPLC system, using an anionexchange column. Several different buffers at the same pH were tested. Tf purified by this method was analyzed by Western blot, followed by immunodetection, as well as with silver staining after SDS PAGE. Its functionality was tested with respect to iron-binding capacity (ferozzine method) and its ability to interact with TFR by immunofluorescent staining. The conformation of purified Tf was analyzed by recording intrinsic fluorescent emmision spectra originating from Trp residues. The method itself is highly reproducible (intra- and interday), easy to perform (only two steps) and fast (under an hour), yielding 98% to 99% pure Tf with all buffers. Purified Tf was shown to have retained its ironbinding capacity, as well as the ability to interact with TFR. Purified Tf also retained its native three-dimensional structure. Described method for the isolation and purification of Tf is fast, simple and highly reproducible, yielding a functional Tf of high purity in its native state while offering the flexibility of using different buffer systems. All of these features make this protocol a method of choice for the isolation and purification of Tf on a semi-preparative scale

Interaction of commonly used antioxidants with major circulating proteins: Spectroscopic and in silico study

Antioxidants, used in human nutrition as food supplements, show poor bioavailability and reducedstability, so in order to utilize their beneficial effects their binding to circulatory proteins is desirable.Besides albumin and IgG, other major proteins in the circulation are fibrinogen (Fib), transferrin(Tf) and alpha-2-macroglobulin (α2M). They are all potential candidates for antioxidant binding.Quercetin, resveratrol and dihydrolipoic acid are among the most commonly used antioxidants inhuman nutrition. The idea of the present study was to investigate whether these three antioxidantsbind to the mentioned plasma proteins. After testing the affinity for ligands, the pair with thehighest association constant was α2M/resveratrol (4.5 x 104 M–1), so this complex was chosento be spectroscopically characterised, and subjected to docking simulation, in order to elucidatethe structure of the binding site(s). The computational simulaitons revealed that α2M possessesfour potential binding sites for resveratrol, and that the formation of hydrogen bonds is crucialfor binding. The binding of resveratrol to α2M leads to mutual protection from oxidative stressand significantly increases hidrosolubility of resveratrol. Both these features serve to increase thebioavailability and bioactivity of resveratrol in the circulation

Optimizacija upotrebe pululanaze iz soja Bacillus paralicheniformis 9945a u hidrolizi skroba

Danas gotovo svi industrijski procesi konverzije skroba u glukozu upotrebljavaju enzimsku hidrolizu umesto tradicionalne kisele metode. Glavna primena pululanaze u industriji šećera je u saharifikaciji skroba, gde se ona koristi za proizvodnju sirupa visokog sadržaja glukoze ili maltoze. U procesu saharifikacije pululanaza se najčešće koristi u kombinaciji sa glukoamilazom ili β-amilazom. Kombinovanjem pululanaze sa drugim amilotičkim enzimima povećava se kvalitet dobijenih šećernih sirupa. Kada se skrob tretira amilazom i pululanazom istovremeno se postiže značajno veća efikasnost reakcije saharifikacije. Prednost korišćenja pululanaze u procesima saharifikacije skroba potiče od činjenice da je skrob koji se koristi u industriji sačinjen od ~80 % amilopektina, polisaharida koji poseduje α-1,6 vezane grane koje amilaza ne može da hidrolizuje, a glukoamilaza hidrolizuje, iako veoma neefikasno. Takođe, smanjuje se procenat izomaltoze nastale dejstvom glukoamilaze. Cilj ovog rada je optimizacija dejstva pululanaze iz soja Bacillus paralicheniformis 9945a na efikasnost hidrolize skroba dejstvom amilaze i glukoamilaze

Targeting NF-κB Signaling: Selected Small Molecules Downregulate Pro-Inflammatory Cytokines in Both Food Allergen and LPS-Induced Inflammation

Although inflammation is primarily a protective response guarding the human body, it can result in a variety of chronic diseases such as allergies, auto-immune, cardiovascular diseases, and cancer. In NF-κB-mediated inflammation, many small molecules and food compounds characterized as nutraceuticals have shown positive effects associated with immunomodulatory properties. We investigated the effects of selected bioactive small molecules, commonly found in food components, vanillyl alcohol (VA) and lauric acid (LA), on different cell lines exposed to pro-inflammatory stimuli, lipopolysaccharide (LPS), and the food allergen actinidin (Act d 1). Pro-inflammatory cytokines were downregulated in response to both VA and LA, and this downregulation was caused by a decrease in the activation of the NF-κB pathway and the translocation of p65, the pathway’s major component. Small nutraceutical molecules, VA and LA, showed not only inhibition of the pro-inflammatory cytokines, but also inhibition of the NF-κB activation, and reduced translocation of the p65 component. The present study may contribute to the therapeutic use of these molecules for various inflammatory diseases, which have in common an increased expression of pro-inflammatory cytokines and NF-κB-mediated inflammation

Interaction of commonly used antioxidants with major circulating proteins: Spectroscopic and in silico study

Antioxidants, used in human nutrition as food supplements, show poor bioavailability and reduced stability, so in order to utilize their beneficial effects their binding to circulatory proteins is desirable. Besides albumin and IgG, other major proteins in the circulation are fibrinogen (Fib), transferrin (Tf) and alpha-2-macroglobulin (α2M). They are all potential candidates for antioxidant binding. Quercetin, resveratrol and dihydrolipoic acid are among the most commonly used antioxidants in human nutrition. The idea of the present study was to investigate whether these three antioxidants bind to the mentioned plasma proteins. After testing the affinity for ligands, the pair with the highest association constant was α2M/resveratrol (4.5 x 104 M–1), so this complex was chosen to be spectroscopically characterised, and subjected to docking simulation, in order to elucidate the structure of the binding site(s). The computational simulaitons revealed that α2M possesses four potential binding sites for resveratrol, and that the formation of hydrogen bonds is crucial for binding. The binding of resveratrol to α2M leads to mutual protection from oxidative stress and significantly increases hidrosolubility of resveratrol. Both these features serve to increase the bioavailability and bioactivity of resveratrol in the circulation

The Insulin-like Growth Factor System and Colorectal Cancer

Insulin-like growth factors (IGFs) are peptides which exert mitogenic, endocrine and cytokine activities. Together with their receptors, binding proteins and associated molecules, they participate in numerous pathophysiological processes, including cancer development. Colorectal cancer (CRC) is a disease with high incidence and mortality rates worldwide, whose etiology usually represents a combination of the environmental and genetic factors. IGFs are most often increased in CRC, enabling excessive autocrine/paracrine stimulation of the cell growth. Overexpression or increased activation/accessibility of IGF receptors is a coinciding step which transmits IGF-related signals. A number of molecules and biochemical mechanisms exert modulatory effects shaping the final outcome of the IGF-stimulated processes, frequently leading to neoplastic transformation in the case of irreparable disbalance. The IGF system and related molecules and pathways which participate in the development of CRC are the focus of this review