The Chemical Determination of Proteins

Experiments of this kind can provide valuable insights into the chemical determinants of protein structure. The results support speculations that ancient proteins might have been constructed from small numbers of amino acids [121 - 123], Nevertheless, the relatively low abundance of active enzymes in the combinatorial libraries... 

The second problem is the possible presence of deoxyhemoglobin in the tissues. Indeed, deoxyhemoglobin could contribute to the transverse relaxation but through a non-linear transverse rate enhancement since for this protein l/T 2 increases quadratically with the magnetic field. Such an effect has only been noticed for blood (51). As a conclusion, the present state-of-the-art allows to claim that MRI provides a qualitative indication and a quantitative estimation of iron accumulation, but is unable to approach the accuracy of the chemical determination of iron content. 

For proteins whose structure is known by means of crystallographic studies, Mossbauer spectroscopy and studies by electron paramagnetic resonance afford an opportunity to determine the detailed electronic configurations, a necessary step towards the chemical basis of protein function. 

The initial preparation of protein-based polymers utilized solution and solid phase peptide chemistry. This made possible the preparation of more than 1,000 polymer compositions. As discussed in Chapter 5, these compositions were studied for determination of their basic properties, for the development of the set of phenomenological axioms for protein engineering and function, and for the demonstration of the basic mechanism that underlies function. In short, it is the chemical synthesis that has allowed development of much of the basic science and the demonstration of the potential of protein-based materials in a timely manner. Mostly because of the historical relevance, but also because of the unique contributions of chemical synthesis to arriving at satisfactory purification of microbially prepared protein-based polymers, a brief description of the chemical synthesis of protein-based polymers is given below. 

The first primary P. structure to be determined was that of insulin in 1953. By the end of 1980, over 700 complete primary P. structures had been reported, using the chemical methods of protein analysis described above. Although these methods are still used, in particular for smaller polypeptides and partial sequences, primary P. sequences are now mostly determined from the structure of the cloned gene encoding the P. in question (see Recombinant DNA technology. Nucleic acid sequencing). In 1995, the... 

Amino acids are simple building-block subunits of proteins, and their side chains determine the higher-order structures and the chemical functions of proteins. The FUV spectral measurements of amino acids especially in gas and solution states are very challenging, because the vapor pressures of amino acids are quite low [66], and the photoabsorption of solvents including water is very intense [7, 14]. Just a few FUV absorption spectra of amino acids were reported in the thin solid films [67,68], the gas states [69,70], and the solution states [71,72]. Actually, the FUV spectra of amino acids in aqueous solutions can be easily obtained by using the ATR method. This section will introduce the FUV spectra of 20 naturally occurring amino acids in pure water, acidic, and basic aqueous solutions in the region from 145 to 300 nm [73]. 

S. B 2ick cn2cn, Mmino Mcid Determination Methods and Techniques, Marcel Dekker, New York, 1968 A. Niederweiser and G. Pataki, eds., Neiv Techniques in Mmino Mcids, Peptide and Protein Mnalysis, Ann Arbor Science Pubhshers, Ann Arbor, Mich., 1971 The Chemical Society of Japan, eds., Neiv Experimental Chemistry Series, Vol. 1 (Biochemistry 1) (in Japanese), Mamzen, Tokyo, Japan, 1978, pp. 141—160. 

The nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of a cell s genetic information. Coded in a cell s DNA is the information that determines the nature of the cell, controls the cell s growth and division, and directs biosynthesis of the enzymes and other proteins required for cellular functions. 

In more recent times chemically defined basal media have been elaborated, on which the growth of various lactic acid bacteria is luxuriant and acid production is near-optimal. The proportions of the nutrients in the basal media have been determined which induce maximum sensitivity of the organisms for the test substance and minimize the stimulatory or inhibitory action of other nutrilites introduced with the test sample. Assay conditions have been provided which permit the attainment of satisfactory precision and accuracy in the determination of amino acids. Experimental techniques have been provided which facilitate the microbiological determination of amino acids. On the whole, microbiological procedures now available for the determination of all the amino acids except hydroxy-proline are convenient, reasonably accurate, and applicable to the assay of purified proteins, food, blood, urine, plant products, and other types of biological materials. On the other hand, it is improbable that any microbiological procedure approaches perfection and it is to be expected that old methods will be improved and new ones proposed by the many investigators interested in this problem. 

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. 

The contractile apparatus may be thought of as the sum of those intracellular components which constitute the machinery of chemomechanical transduction. It is the set of proteins which convert the chemical energy of the terminal phosphate ester bond of ATP into mechanical work. The structure of the contractile apparatus is determined by the connections between the various protein molecules via specific binding sites or, in a minority of cases, via labile covalent linkages. The kinetics of the contractile machinery are determined by the regulation of changes in these connections. 

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