Ammonium sulfate precipitation combined with liquid chromatography is sufficient for purification of bovine serum albumin that is suitable for most routine laboratory applications

The use of bovine serum albumin (BSA) in routine biochemical assays such as restriction enzyme digestion and immunodetection is plagued largely by two common contaminants, DNase and immunoglobulins G (IgGs). Acetylation of BSA to inactivate DNase limits its use as a protein standard due to interference with color development in assays such as Lowry’s. In spite of the availability of inexpensive BSA, its purification involves several cumbersome and time-consuming steps. In this work, we employed a modified strategy of ammonium sulfate precipitation coupled with liquid chromatography to purify BSA that is free of DNase and IgGs. Purified BSA tested negative for the presence of DNase and IgGs using DNase and immunodetection assays respectively. We conclude that carefully controlled ammonium sulfate precipitation and liquid chromatography techniques are sufficient to purify BSA suitable for most routine laboratory applications. This purification strategy can yield more than 40 g BSA per liter of serum

Sodium Nitrite Alone Protects the Brain o _i_ Microsomal Ca -ATPase Against Potassium Cyanide-induced Neurotoxicity In Rats

The effect of a short-term oral administration of potassium cyanide (KCN) (200 ppm in diet) with or without sodium nitrite (NaNO2) pretreatment on rat brain microsomal Ca2* ATPase was investigated. The specific activity value of the enzyme significantly decreased (p\u3c0.05) by 50% compared with control and by 63% for KCN-treated rats compared with KCN-treated rats pretreated with NaNO;,. There was no significant difference at the h = 0.05 level between the values obtained for the control and KCN-treated rats pretreated with NaNO,. These results show both that feeding lowers brain microsomal Ca2f-ATPase activity and that NaNO, has a protective role (antidote function) in that respect

Reversal of Sodium Arsenite Inhibition of Rat Liver Microsomal Ca2+ Pumping ATPase by Vitamin C

Sodium arsenite (NaAsO2), at 10% of its median lethal dose, was administered to rats with and without vitamin C pretreatment. Liver microsomal fraction was isolated and the activity of Ca2+-ATPase was assayed. Sodium arsenite was found to inhibit the activity of the liver microsomal Ca2+-ATPase to 50% to that of control rats. The specific activity of the enzyme in rats administered sodium arsenite with vitamin C pretreatment was not significantly different from that of control rats

Iron-Induced Oxidative Stress in Erythrocyte Membranes of Non-Insulin-Dependent Diabetic Nigerians

The presence of higher level of endogenous free radical reaction products in the erythrocyte ghost membrane (EGM) of Non-insulin-dependent diabetes mellitus (NIDDM) subjects compared with that of normal healthy controls has been demonstrated. The EGMs of NIDDM subjects were also shown to be more susceptible to exogenously generated oxidative stress than those of normal healthy individuals. The decreased level of reactive thiol groups in the EGM of NIDDM individuals supported this observation. We propose that the presence of significant levels of non-heme iron in the EGM of NIDDM subjects is an indication of the potential for iron-catalysed production of hydroxy and other toxic radicals which could cause continuous oxidative stress and tissue damage. Oxygen free radicals could therefore be responsible for most of the erythrocyte abnormalities associated with non-insulin-dependent diabetes and could indeed be intimately involved in the mechanism of tissue damage in diabetic complications

Design of a robust undergraduate biochemistry laboratory course based on a modified and expanded bovine serum albumin purification scheme

The ASBMB curriculum for an undergraduate degree recommends a set of skills that can be acquired only through laboratory courses and research experience. Based on a previously reported purification scheme for BSA (Odunuga and Shazhko, 2013), we designed a robust, reproducible, cost-effective, safe and enquiry-based undergraduate biochemistry laboratory course that encompasses many of the skill-sets recommend-ed in the ASBMB curriculum. Our work not only modi-fied certain steps in the scheme, it also included addi-tional steps to enhance student learning and skill ac-quisition. Salt precipitation, ion exchange and size ex-clusion chromatography were employed by students to purify BSA from cow plasma. Presence of major contaminants of BSA purification, IgGs and nucleases, were tested in the purified sample by western blotting and DNase I assay respectively. The DNase as-say step provides an opportunity for students to learn basic molecular biology techniques such as plasmid isolation and restriction-enzyme digestion. One major addition to the purification process is the bromocre-sol green-BSA complex assay to precisely quantitate BSA at each step and generate a purification table. Comparison of sequences and other parameters of al-bumin proteins from common animals provides a bio-informatics twist to student experience. Abundance of albumin from the plasmas of common animals, cow, pig and chicken, allows for variation in the de-sign of the course, and students can work in groups or individually. The course could be designed as a half-semester or full-semester biochemistry laboratory module

The use of a quartz crystal microbalance with dissipation for the measurement of protein–protein interactions: a qualitative and quantitative analysis of the interactions between molecular chaperones

Biotechnology research and innovation depends on the ability to understand the molecular mechanisms of biological processes such as protein–protein and protein–ligand interactions. Surface plasmon resonance (SPR) spectroscopy is now well established as a quantitative technique for monitoring biomolecular interactions. In this study, we examined the recently developed quartz crystal microbalance with dissipation (QCM-D) method as an alternative to SPR spectroscopy to investigate protein–protein interactions, in particular, for chaperone–co-chaperone interactions. In mammalian cells, the Hsp70/Hsp90 organizing protein (Hop) is a co-chaperone required for the association of the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). The objective of this research was to characterize qualitatively and quantitatively the interaction of Hsp70 with Hop. A truncated version of Hop consisting of only the C-terminal region and lacking the Hsp70-binding domain (GST-C-Hop) was used as a non-Hsp70- binding control. Immobilized GST-Hop was found to bind Hsp70 successfully, displaying a QCM-D response consistent with formation of a complex that became slightly more flexible as the concentration of bound Hsp70 increased. GST-C-Hop did not bind to Hsp70, thereby validating the specificity of the GST-Hop interaction with Hsp70. The kinetics of the interaction was followed at different concentrations of Hsp70, and an apparent thermodynamic dissociation constant (KD value) in the micromolar range was determined that correlated well with the value derived previously using SPR. This study represents a proof-of-principle that QCM-D can be applied to the analysis of chaperone–co-chaperone interactions. The economic and technical accessibility of QCM-D makes it a valuable tool for analyses of chaperone interactions, and protein– protein interactions in general

Mechanical function and Biophysical Properties of the REJ region of Polycystin-1

Mutations of Polycystin-1 (PC1) account for 85% Autosomal dominant polycystic kidney disease (ADPKD), which is the most common life-threatening inherited disease worldwide. PC1 has been implicated to be involved in renal tubule and kidney morphogenesis as a mechanosensor and transduce the signals into cellular response. Most domains of the long PC1\u27s ectodomain of are mechanical stable Ig-like motifs and may function as effective force transmitters to regulate the multi-function properties of PC1. The REJ region is a major component of PC1s ectodomain (30% or ∼1000 aa); however its structure and function remains unknown. Here we used protein engineering in combination with single-molecule AFM and circular dichroism (CD) techniques to elucidate the structure and mechanical properties of this region. Our studies indicates that the REJ region has complex mechanical properties. Stretching a protein construct which includes four PKD Ig-like domains and the complete REJ region, resulted in saw-tooth patterns with 3-10 force peaks with a wide range of unfolding forces of 50-250 pN, suggesting that the extra force peaks must originate from the REJ region. We also made several REJ constructs (I27)3-REJ FN4-(I27)2 and (I27)3-REJ FN3,4-(I27)2 and expressed them in bacteria and insect cells. Stretching these constructs generated peaks characteristic of the unfolding of titin I27 as well as other more complex unfolding events which we attribute to the unfolding of REJ domains. The complexity of the REJ domain unfolding force patterns suggests that these domains may have unfolding intermediates. These results support the hypothesis that PC1 is a mechano-transducer with a novel molecular architecture and elastic properties well-suited for sensing and transmitting distinct mechanical signals with a wide range of strengths