Determination of Proteins

Although each acid-base titrimetric method has its own unique considerations, the following description of the determination of protein in bread provides an instructive example of a typical procedure. 

Orstan, A. Wojcik, J. F. Spectroscopic Determination of Protein-Ligand Binding Constants, /. Chem. Educ. 1987, 64, 814-816. 

TF Flavel. An evaluation of computational strategies for use m the determination of protein structure from distance constraints obtained by nuclear magnetic resonance. Prog Biophys Mol Biol 56 43, 1991. 

To gain the most predictive utility as well as conceptual understanding from the sequence and structure data available, careful statistical analysis will be required. The statistical methods needed must be robust to the variation in amounts and quality of data in different protein families and for structural features. They must be updatable as new data become available. And they should help us generate as much understanding of the determinants of protein sequence, structure, dynamics, and functional relationships as possible. 

A very narrow window produces monochromatic radiation that is still several orders of magnitude more intense than the beam from conventional rotating anode x-ray sources. Sucb beams allow crystallographers to record diffraction patterns from very small crystals of the order of 50 micrometers or smaller. In addition, the diffraction pattern extends to higher resolution and consequently more accurate structural details are obtained as described later in this chapter. The availability and use of such beams have increased enormously in recent years and have greatly facilitated the x-ray determination of protein structures. 

Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595 nm when the reagent binds to the protein. A 20 mg/1 bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for determination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

Dalluge, J. J. Mass spectrometry for direct determination of proteins in cells Applications in bitechnology and microbiology Fresenius. J. Anal. Chem. 2000,366, 701-711. 

Determination of protein secondary structure has long been a major application of optical spectroscopic studies of biopolymers (Fasman, 1996 Havel, 1996 Mantsch and Chapman, 1996). These efforts have primarily sought to determine the average fractional amount of overall secondary structure, typically represented as helix and sheet contributions, which comprise the extended, coherent structural elements in well-structured proteins. In some cases further interpretations in terms of turns and specific helix and sheet segment types have developed. Only more limited applications of optical spectra to determination of tertiary structure have appeared, and these normally have used fluorescence or near-UV electronic circular dichroism (ECD) of aromatic residues to sense a change in the fold (Haas, 1995 Woody and Dunker, 1996). 

Meadows F, Narayanan N, Patonay G (2000) Determination of protein-dye association by near infrared fluorescence-detected circular dichroism. Talanta 50 1149-1155... 

Fig. 2.9. The thermodynamic cycle used for the determination of protein-ligand relative binding free energies. Instead of carrying the horizontal transformations one can mutate the ligand in the free state - i.e., the left, vertical alchemical transformation , and in the bound state -i.e., the right, vertical alchemical transformation. This yields the difference in the binding free energies. AA I, jI,I j T. binding A mutation mutation...

Ahn, B., Rhee, S.G., and Stadtman, E.R. (1987) Use of fluorescein hydrazide and fluorescein thiosemicar-bazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Anal. Biocbem. 161, 245-257. 

Dearman, R.J. and Kimber, I., Determination of protein allergenicity Studies in mice. Toxicol. Lett., 120, 181, 2001. 

T. B. Fanner and R. M. Caprioli. Determination of Protein-Protein Interactions by Matrix-Aassisted Laser Desorption/Ionization Mass Spectrometry. J. Mass Spectrom., 33(1998) 697-704. 

Electrophoresis has been applied for decades in analytical chemistry. It is still the method of choice for the determination of proteins and nucleic acids. The technique of CE is classified in three main groups of methods [1] ... 

Experiment 12 Determination of Protein in Macaroni by the Kjeldahl Method... 

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