Tryptophan accuracy

Sample Amount Distributed Positive Accuracy Cysteine Accuracy Tryptophan Accuracy... 

The constitution of indole and skatole had been proved by synthesis, but that of the other two compounds had not been determined, and Nencki s formulae for them were accepted. Investigations by Ellinger and Gentzen in 1903, who found that in the large intestine indole was formed in large amounts from tryptophane, but skatole only in small amounts, and that skatole only gave traces of indole under the same conditions led Ellinger to doubt the accuracy of Nencki s formulae for skatoleacetic and skatolecarboxylic acids, more especially as Wislicenus and Arnold s skatolecarboxylic acid, which was synthesised from pro-pionyl formic acid phenylhydrazone had the formula... 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

Direct Photometric Methods. Absorption of ultraviolet (UV) light at 200 to 225 nm and 270 to 290 nm is used to measure the protein content of biological samples. Absorption of UV light at 280 nm depends chiefly on the aromatic rings of tyrosine and tiyptophan at pH 8. Accuracy and specificity suffer from an uneven distribution of these amino acids among individual proteins in a mixture and from the presence in body fluids of free tyrosine and tryptophan, uric... 

It is evident from the published data and from data privately collected that considerable variations exist between the values of these constants as determined in different laboratories. This applies particularly to tryptophan. The question is discussed separately for each amino acid but it is relevant here to comment generally on the accuracy of spectrophotometric methods. 

Several proteins can be cited (see Table VII) in which the agreement between the spectrophotometric figures for tyrosine and tryptophan and the analytical data obtained by other methods is sufficiently close to justify the conclusion that in these proteins there cannot be any significant contribution to the absorption spectrum by the peptide fabric, in the 2700-3100 A. region. Any such contribution would have seriously affected the accuracy of the spectrophotometric analysis. 

There are several significant advantages to the laser source over the more conventional flash lamp. The laser source is more intense. Its pulse width is near 5 psec, as compared for 2000 psec for a flash lamp. And finally, the repetition rate can be much higher, typically 800 kHz to 4 MHz. Because of all these factors it is possible to rapidly acquire data to a much higher level of statistical accuracy than with a flash lamp. For example, a recent paper by Small and co-workers describes a multi-component resolution of a histone, which contains a single tyrosine residue [31]. Because of the substantial increases in resolution, the laser sources are becoming more widely used in the biochemical applications of fluorescence, as illustrated by recent studies of the tryptophan emission from phospholipase Aj [44] and hemoglobin [45]. 

Recently magnetic circular dichroism (MCD) has been used to determine the tryptophan content of proteins 190). The intense positive band near 293 nm, uniquely characteristic of tryptophan, allows direct and quantitative measurement of this amino acid with high sensitivity and accuracy in the intact protein (see Section V.2.I., for a more detailed discussion of the method). However, although the technique seems to be very promising, it has the disadvantage of requiring highly specialized equipment. 

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