Protein photometric determination

Among the analytical methods presently used for the characterization of natural and synthetic peptides and proteins, the primary value of amino acid analysis is the determination of absolute peptide and protein content in solids and solutions and the quantitation of their amino acid composition and stoichiometry. It involves two steps, i.e. complete hydrolysis of peptides and proteins, followed by photometric determination of the released amino adds. The steps are laborious and time-consuming, and there is a continuous need for improvement of the techniques to increase precision and sensitivity. 

Active immobilized enzyme spectro-photometric determination of bound protein... 

In ester synthesis and exchange reactions, as well as in hydrolysis re tions induced by PEG-lipase in hydrophobic media, the existence of a trace amount of water in the reaction system was most important in terms of the reactions proceeding. Matsushima et al. [67] carried out a kinetics study of PEG-lipase in transparent benzene solution to estimate the value of water, one of the substrates of lipase in the ester hydrolytic reaction. Indoxyl acetate was hydrolyzed by PEG-lipase to form acetic acid and 3-hydroxyindole, which was photometrically determined. A double-reciprocal plot of the velocity of the indoxyl acetate hydrolysis against water concentration at a given concentration of indoxyl acetate indicated that the hydrolysis took place as a double-displacement reaction (ping-pong reaction). The apparent Michaelis-Menten constant of water and the maximum velocity were calculated to be = 7 X 10 M and Vmax = 4700 xmol/min/mg of protein, respectively. 

Unique methods based on new principles have been developed within the past 10 years. Threonine (27,28,249) is oxidized by lead tetraacetate or periodic acid to acetaldehyde, which is determined by photometric analysis of its p-hydroxydiphenyl complex or iodometric titration of its combined bisulfite. Serine is oxidized similarly to formaldehyde, which is determined gravimetrically (207) as its dimedon (5,5-dimethyldihydro-resorcinol) derivative or photometric analysis (31) of the complex formed with Eegriwe s reagent (l,8-dihydroxynaphthalene-3,5-disulfonic acid). It appears that the data obtained for threonine and serine in various proteins by these oxidation procedures are reasonably accurate. [Block and Bolling (26) have given data on the threonine and serine content of various proteins. ]... 

The results obtained with ISEs have been compared several times with those of other methods. When the determination of calcium using the Orion SS-20 analyser was tested, it was found that the results in heparinized whole blood and serum were sufficiently precise and subject to negligible interference from K and Mg ([82]), but that it is necessary to correct for the sodium error, as the ionic strength is adjusted with a sodium salt [82], and that a systematic error appears in the presence of colloids and cells due to complexa-tion and variations in the liquid-junction potential [76]. Determination of sodium and potassium with ISEs is comparable with flame photometric estimation [39, 113, 116] or is even more precise [165], but the values obtained with ISEs in serum are somewhat higher than those from flame photometry and most others methods [3, 25, 27, 113, 116]. This phenomenon is called pseudohyponatremia. It is caused by the fact that the samples are not diluted in ISE measurement, whereas in other methods dilution occurs before and during the measurement. On dilution, part of the water in serum is replaced by lipids and partially soluble serum proteins in samples with abnormally increased level of lipids and/or proteins. 

Besides being presently the most efficient method for site-directed interchain disulfide bridging of two different cysteine peptides, this procedure is also recommended for a controlled peptide-protein conjugation by reacting Npys-protected cysteine peptides with properly thiol-functionalized protein carriers. 1761 In this conjugation procedure the amount of peptide grafted to the protein is quantitatively determined by measuring spectro-photometrically at 430 nm the amount of 3-nitropyridine-2(l//)-thione liberated in the reaction. An example of this approach is outlined in Scheme 18. 

The Folin-Ciocalteau Assay of Protein Concentration. The Folin-Ciocalteau assay is one of the most sensitive and most commonly used assays to determine protein concentration (sensitive to about 10 /rg/m I protein). This procedure employs two color-forming reactions to assay protein concentration photometrically. In the first reaction (a biuret reaction), compounds with two or more peptide bonds form a dark blue-purple color in the presence of alkaline copper salts. In the second reaction, tryptophan and tyrosine side chains react with the Folin solution to produce cuprous ions. This reaction is most efficient under basic condi-... 

Hartree, E.F. (1972) Determination of Protein A Modification of the Lowry Method that Gives a Linear Photometric Response, Anal. Biochem. 48,422—427. 

Another example of on-line monitoring of enzyme activities was given by Kunnecke et al. [88], when a FIA-system was used for the determination of enzyme activities during protein purification by fast protein liquid chromatography (FPLC). Photometric assays for four different oxidases were established in a FIA-system extending the linear range by the so-called zone sampling method. The FIA-device was coupled to the FPLC unit behind a... 

There are several recorded determinations of the absorption curves of the aromatic amino-acids. Most of these were obtained with photographic methods of spectrophotometry which have been superceded by more accurate photoelectric methods. It will be shown that in the spectrophotometric analysis of tyrosine and tryptophan in proteins, the photometric error is magnified in the final estimate of tyrosine and tryptophan contents. This fact is inevitably bound up with the form of the equations of mixture analysis. It is therefore important that the absorption constants be measured as accurately as possible. 

Hartree EF (1982) Determination of proteins a modification of the Lowry method that gives a linear photometric response. Anal Biochem 48 422-427... 

Tenhunen [167] quotes a simple TLC-procedure for separation of bile pigments from human bile. The diazotised pigments were separated on silica gel G layers with the solvent butanone-propionic acid-water (60 + 15 + 15) bilirubin sulphate, bilirubin glucuronide, bilirubin monoglucuronide, free bilirubin and biliverdin were found in this order of increasing i /-value and were eluted and determined quantitatively by a photometric method. The glucuronic acid content can be determined also after acid hydrolysis. In serum, proteins must be precipitated beforehand. 

It must be evident to the reader that more work is needed to determine the amino acids other than tyrosine in proteins which react with iodine. Since iodine has a powerful effect on some absorbing groups, spectro-photometric analyses coupled with variations of pH and other factors might prove very useful. In the case of tyrosine, the ionization constants, or pKs of phenolic groups of the iodo derivatives have been determined spectrophotoroetrically (75, 307). For tyrosine (75, 308), mono iodo-tyrosine (308), and diiodotyrosine (75, 308) the pKs are 10.1, 8.2 and 6.4 respectively. 

The reaction is the basis of various methods for the determination of amino acids since it is possible to measure (1) the CO2 produced, (2) the NH3 produced and (3) the colour intensity obtained when the liberated ammonia reacts with a further molecule of ninhydrin to produce a purple compound which can be assayed photometrically. This is the method by which amino acids are estimated using an amino acid analyser. The imino acids (proline and hydroxyproline) give yellow products instead of a purple one. Amino acids give a strong reaction with ninhydrin, but proteins and polypeptides, which contain far fewer free amino groups, give a much weaker reaction. 

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