Bovine serum albumin spectroscopy

This experiment provides a nice example of the application of spectroscopy to biochemistry. After presenting the basic theory for the spectroscopic treatment of protein-ligand interactions, a procedure for characterizing the binding of methyl orange to bovine serum albumin is described. 

The interaction between 4-(4-hydroxybut-2-ynyloxy)-3-(phenylsulfonyl)-l,2,5-oxadiazole-2-oxide 16 and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV-Vis absorption spectroscopy. The results indicate that molecules 16 bind with BSA forming 1 1 complex. Thermodynamic parameters, such as AH, AG, and A.Y, were calculated. The results indicate that the binding reaction is mainly entropy driven and hydrophobic forces play a major role in this reaction <2006CHJ1050>. 

The mechanism of adsorption of drugs by montmorillonite has been studied using infrared (IR) spectroscopy and XPD [68,69]. X-ray diffractometry and a host of other techniques were used to study the interaction of bovine serum albumin with the surface of a microcrystalline aluminum oxide hydroxide compound [70]. 

Aminophthalate anion Atmospheric pressure active nitrogen Analyte pulse perturbation-chemiluminescence spectroscopy Arthromyces rasomus peroxidase Ascorbic acid Adenosine triphosphate Avalanche photodiode 5-Bromo-4-chloro-3-indolyl 2,6-Di-t< r/-bu(yl-4-mclhyl phenol Bioluminescence Polyoxyethylene (23) dodecanol Bovine serum albumin Critical micelle concentration Calf alkaline phosphatase Continuous-addition-of-reagent Continuous-addition-of-reagent chemiluminescence spectroscopy Catecholamines Catechol... 

Abbreviations BSA, bovine serum albumin PBS, phosphate buffered saline PMFS, phase-modulation fluorescence spectroscopy TCSPC, lime-correlated single photon counting. 

Morrissey 53) used transmission infrared spectroscopy to study protein adsorption onto silica particles in a heavy water (DzO) buffer. By observing the shift in the amide I absorption band, he could deduce the fraction of protein carbonyl groups involved in bonding to the silica surface. He found that bovine IgG had a bound fraction of 0.20 at low bulk solution concentrations, but only about 0.02 at high solution concentrations. However, neither prothrombin nor bovine serum albumin exhibited a change in bound fraction with concentration. Parallel experiments with flat silica plates using ellipsometry showed that the IgG-adsorbed layers had an optical thickness of 140 A and a surface concentration of 1.7 mg/m2 at low bulk solution concentration — in concentrated solutions the surface amount was 3.4 mg/m2 with a thickness of 320 A (Fig. 17). 

Frictional Force Microscopy Fourier-transform infrared spectroscopy scanning electron microscope surface force apparatus Secondary ion mass spectroscopy scanning tunneling microscope X-ray photoelectron spectroscopy bovine serum albumin immunoglobulin G... 

Markelz AG, Roitberg A, Heilweil EJ. Pulsed terahertz spectroscopy of DNA, bovine serum albumin and collagen between 0.1 and 2.0 THz. Chem Phys Lett. 2000 320(1-2) 42-48. 

They confirm that 0.1 M potassium chloride lowers the pH at which the expansion occurs and yet show that it does not greatly affect the isomerization near pH 4. Since the isomerization is undetectable at 589 m i, the wavelength used by Kauzmann, the optical rotation at this point will not parallel changes as indicated by other techniques. The volume contraction which continues in 0.15 M potassium chloride near pH 4 may therefore reflect a slight unfolding during isomerization, a possibility that is in harmony both with the concomitant exposure of about 20% of the tyrosyl residues in bovine serum albumin as measured by difference spectroscopy and the small loss in helical content. 

The reaction of Cr(VI) with bovine serum albumin (BSA) at pH 7.4 results in the formation of Cr(V) species (detected by EPR spectroscopy, gi o = 1.9790) (411). The reported giso value is characteristic of hve-coordinate Cr -oxo complexes with 1,2-diolato coordination (I in Chart 1 Section II.A.2) (12, 18, 27). This complex, however, may be an artifact due to the presence of trace carbohydrates in BSA, as no signihcant EPR signals were observed during the reactions of high purity BSA with Cr(VI) (412). Photocatalyzed reactions of Cr(VI) with amino acids (Gly or Ala) or with small peptides (Gly or Ala , n = 3-5) in methanol solutions led to the formation of relatively stable Cr -amino acid(peptide) complexes (such as II in Chart 1), characterized by EPR spectroscopy and electrospray mass spectrometry (22, 23, 34). An EPR silent Cr -Ala dimer (III in Chart 1) has been isolated and characterized by XAFS spectroscopy this is the first structurally characterized Cr -amino acid complex... 

Figure 5. Hydrolysis of fluorogenic substrate 1 by HTV protease at 37 C as monitored by steady state fluorescence spectroscopy (Xex+340, A m=490). The reaction was carried out with 10 pM substrate at pH 4.7 in a buffer solution containing 0.1 M NaOAc, 1.0 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% DMSO and 1 mg/mL bovine serum albumin. The arrow indicates the point of addition of HTV protease to a final concentration of 35 nM. Product analysis was carried out by HPLC, mass spectrum and fluorescence lifetime. Inset The initial phase of the hychx)lysis reaction used for rate determinations.

Chemical modifications by derivitizing the surfaces of mineral fiber silicates have revealed alterations in reactivity in in vitro cell toxicity studies, suggesting a multifactorial character of particle/cell interactions [171]. In addition, the adsorption of bovine serum albumin onto asbestos fibers has been shown by infrared spectroscopy and NMR to be mediated by O—H—N hydrogen bonds [172]. In view of the various hypotheses put forward in the bulk biochemical studies and the apparent surface-oriented origins of these reactions, it is important to examine the features of the silicate/cell interfaces and surfaces. Herein we present a typical case of the interaction between a related alumino-silicate (i.e., cummingtonite) and bioorganic cell interaction. 

McCabe and Fisher (1965) have measured the excluded volume of protein molecules by means of differential spectroscopy in the near-infrared region. The excluded specific volumes of bovine serum albumin and lysozyme were found to be 0.74 ml/g and 0.68 ml/g, respectively, values which are close to the values of apparent specific volume reported in the literature (Dayhoff et al., 1952 Sophianopoulos et al, 1962). The authors suggested that excluded specific volume as measured by their method is closely associated with the partial specific volume of the protein molecule. 

Electrochemistry can be coupled with other physical methods such as fluorescence spectroscopy. An XO-based electrochemiluminescent biosensor for hypoxanthine has been reported. The enzyme was immobilized in a carbon paste electrode with bovine serum albumin cross-linked with glutar-aldehyde. The working principle of the biosensor is illustrated in Scheme 5.6. As already shown (eqn (5.3a)), H2O2 is produced by the catalytic reaction between hypoxanthine and XO immobilized on the electrode surface. In an alkaline or neutral solution, luminol is electrochemically oxidized to a compound that reacts spontaneously with H2O2 to generate chemiluminescent luminol and the ensuing luminescence was used to quantify the amount of hypoxanthine present. 

Investigation of complex biological samples, such as the analysis of bovine serum albumin, could be best achieved by employing the high resolving power of multidimensional fluorescence spectroscopy. An isometric projection will allow for a simultaneous observation of all the data points in each of the scans. In addition to a more pictorial representation of the... 

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