Identification of the Protein Glycation Sites in Human Myoglobin as Rapidly Induced by d-Ribose

Protein glycation is an important protein post-translational modification and is one of the main pathogenesis of diabetic angiopathy. Other than glycated hemoglobin, the protein glycation of other globins such as myoglobin (Mb) is less studied. The protein glycation of human Mb with ribose has not been reported, and the glycation sites in the Mb remain unknown. This article reports that d-ribose undergoes rapid protein glycation of human myoglobin (HMb) at lysine residues (K34, K87, K56, and K147) on the protein surface, as identified by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Moreover, glycation by d-ribose at these sites slightly decreased the rate of the met heme (FeIII) in reaction with H2O2 to form a ferryl heme (FeIV=O). This study provides valuable insight into the protein glycation by d-ribose and provides a foundation for studying the structure and function of glycated heme proteins.

DNA Finger-Printing: Current Scenario and Future

Linearly arranged chemical structure in chromosome is known as DNA. It is a double helix made up of two strands of genetic material spiraled around each other. Each strand has a sequence of bases. There are four types of basis namely adenine, guanine, cytosine and thiamine which are very unique to each individual just like their actual fingerprint. The nitrogen base adenine always binds with thymine and cytosine also always binds with guanine. Thus the DNA profiling unique to each individual is collectively known as DNA fingerprinting. DNA determines individuality or uniqueness of the each human being except in uniovular twins. The chances of complete similarity are one in 30 billion to 300 billion i.e. half the population of world. The technique of DNA fingerprinting was first developed by Dr. Alec Jeffery’s from Britain in 1984. He discovered a minisatellite region close to the human myoglobin gene. He isolated this sequence and used it as a probe to investigate human DNA. He found that the minisatellite probe result was a complex band pattern for each individual. In India, initially it was done at CCMB, Hyderabad by Dr. Lalji Singh. Now there are various centers where DNA fingerprinting is carried out. In Maharashtra it is carried out at Sate Forensic Science Laboratory, Vidya Nagar, Kalina, Mumbai – 400 098 (Phone 022–26670755). Using this technique FBI formally concluded the participation of Mr. Bill Clinton in Monica Lewyninskey case. In India more than 79 cases have been solved by using this technique including important case of Dhanu and Shivarasan alleged assailant of Late Priminister Shr. Rajiv Gandhi, Tandori case, Madhumati murder case etc.

Affimer-Based Europium Chelates Allow Sensitive Optical Biosensing in a Range of Human Disease Biomarkers

The protein biomarker measurement has been well-established using ELISA (enzyme-linked immunosorbent assay), which offers good sensitivity and specificity, but remains slow and expensive. Certain clinical conditions, where rapid measurement or immediate confirmation of a biomarker is paramount for treatment, necessitate more rapid analysis. Biosensors offer the prospect of reagent-less, processing-free measurements at the patient’s bedside. Here, we report a platform for biosensing based on chelated Eu3+ against a range of proteins including biomarkers of cardiac injury (human myoglobin), stroke (glial fibrillary acidic protein (GFAP)), inflammation (C-reactive protein (CRP)) and colorectal cancer (carcinoembryonic antigen (CEA)). The Eu3+ ions are chelated by modified synthetic binding proteins (Affimers), which offer an alternative targeting strategy to existing antibodies. The fluorescence characteristics of the Eu3+ complex with modified Affimers against human myoglobin, GFAP, CRP and CEA were measured in human serum using λex = 395 nm, λem = 590 and 615 nm. The Eu3+-Affimer based complex allowed sensitive detection of human myoglobin, GFAP, CRP and CEA proteins as low as 100 fM in (100-fold) diluted human serum samples. The unique dependence on Eu3+ fluorescence in the visible region (590 and 615 nm) was exploited in this study to allow rapid measurement of the analyte concentration, with measurements in 2 to 3 min. These data demonstrate that the Affimer based Eu3+ complexes can function as nanobiosensors with potential analytical and diagnostic applications.

Protective Potential of Uric Acid, Folic Acid, Glutathione and Ascorbic Acid against the Formation of Toxic Met-Myoglobin

Background: Myoglobin is an oxygen binding protein and its dysfunction has been associated with the pathology of several human disorders. This study was undertaken to investigation the role of hydrogen peroxide (H2O2) in the formation of met-myoglobin and the protective potential of four different reductants such as uric acid, folic acid, glutathione and ascorbic acid were also tested against met-myoglobin formation. Methods: Human myoglobin was treated with H2O2 in-vitro in order to prepare met-myoglobin. The generation of metmyoglobin was confirmed by UV-visible spectroscopy and its stability was analysed by the treatment of human myoglobin with H2O2 at varying pH or time. High performance liquid chromatography (HPLC) was used to determine the oxidatively modified heme products in met-myoglobin. Spectroscopic analysis was used to identify the protective potential of uric acid, folic acid, glutathione and ascorbic acid against the formation of met-myoglobin. Results: The novel data of this study showed that H2O2 induced extensive damage of myoglobin but the treatment with uric acid, folic acid, glutathione or ascorbic acid provides protection of myoglobin against H2O2 induced oxidative damaged. The study apparently proved the protective potential of all these compounds against the toxicity produced by H2O2. Conclusion: This is the first study that shows uric acid, folic acid, glutathione and ascorbic acid provide protection against the generation of toxic met-myoglobin and might be used therapeutically to modify the blood conditions in order to prevent the progression of human disorders associated with myoglobin dysfuntion.

Plant based production of myoglobin - a novel source of the muscle heme-protein

Myoglobin is a heme-protein in the muscle of vertebrates with important functions in the oxygenation of tissues and as a regulator in nitric oxide signaling. Myoglobin from many species is also an important nutritional source of bioavailable iron. In this study, we have successfully produced human myoglobin in the leaves of Nicotiana benthamiana by transient expression using a viral vector delivered by Agrobacterium tumefaciens. Analyses confirmed that heme was incorporated and the protein was functional, with observed properties consistent with those of native myoglobins. A relatively high degree of purity could be achieved with low cost methods. The results show the high potential of plants as a production platform for heme proteins, a group of proteins of interest for iron nutrition applications and possible future pharmaceutical development.