Spectrophotometric analysis protein

An area worthy of study is the development of systems of increasing sample throughput beyond the single column operation. Scott has introduced a prototype multicolumn system based on the centrifugal analyzer principle (53). In this set-up a series of LC colimns is rotated on a disc, with sample delivery at the center of the disc and elution and spectrophotometric analysis on the outside. He has suggested using affinity columns for rapid serum protein analysis by this approach. Of course, other principles, such as segmented flow, could be envisioned in an automated LC system as well. Undoubtedly, we can expect to see the availability of such systems in the next few years. 

Appropriate control experiments were carried out, when both modified and unreacted collagen membrane was immersed in buffer solution (0.02M phosphate buffer, pH 7.0) and stored at 4°C for twenty four hours. Spectrophotometric analysis of the buffer media showed no absorbance from impurities and/or carrier protein at 280nm. Thus, the decrease in absorbance at 280 nm was a direct measure of the binding or sorption of enzyme to the collagen matrix. 

The identification of reactive or chemically modified residues of proteins is often extremely important for the characterization of proteins and their activity. Peptide mapping in conjunction with Edman sequencing and/or mass spectrophotometric analysis has been the method of choice to accomplish this characterization. However, this approach alone may not be sufficient or optimal for every situation as was the case when trying to identify the affinity ligand attachment sites on the B-chain of blocked ricin (Lambert et al., 1991a). 

End-group analysis of proteins. The reagent is used in the same way that phenyl-isocyanate is used (which see), and has the advantage for spectrophotometric analysis that the naphthyl derivatives have a maximum at 222 mp. of high intensity. ... 

Prior to GC or spectrophotometric analysis, hydrazine and dimethylhydrazines must be separated from the biological sample matrix and derivatives of the compounds must be prepared. Separation is usually effected by precipitation of residual protein with acid and extraction of interfering lipids with methylene chloride (Alvarez de Laviada et al. 1987 Preece et al. 1992a Reynolds and Thomas 1965 Timbrell and Harland 1979). Hydrazine and 1,1-dimethylhydrazine, but not 1,2-dimethylhydrazine, may then be derivatized with an aldehyde such as pentafluorobenzaldehyde or / -dimethylaminobenzaldehyde. 

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. 

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. 

This criticism does not apply to measurement of the diffusion constant of molecular proteins by the porous disk method if chemical or spectrophotometric analysis is employed under proper conditions. 

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

COMPOUNDING OF ERRORS. Data collected in an experiment seldom involves a single operation, a single adjustment, or a single experimental determination. For example, in studies of an enzyme-catalyzed reaction, one must separately prepare stock solutions of enzyme and substrate, one must then mix these and other components to arrive at desired assay concentrations, followed by spectrophotometric determinations of reaction rates. A Lowry determination of protein or enzyme concentration has its own error, as does the spectrophotometric determination of ATP that is based on a known molar absorptivity. All operations are subject to error, and the error for the entire set of operations performed in the course of an experiment is said to involve the compounding of errors. In some circumstances, the experimenter may want to conduct an error analysis to assess the contributions of statistical uncertainties arising in component operations to the error of the entire set of operations. Knowledge of standard deviations from component operations can also be utilized to estimate the overall experimental error. 

Preparation of Con A Derivatives. Ca -Zn -Con A was obtained from Miles-Yeda. Ca +-Mn -Con A was prepared as previously described (6). Atomic absorption analysis of these two Con A preparations showed essentially equal amounts of the transition metal ion and calcium ions. Sample solutions (0.6 ml) contained Con A at the appropriate concentration in pH 5.60, 0.1N potassium acetate buffer, y = 1.0 in potassium chloride. The final protein concentration was determined spectrophotometrically using = 12.4 at 280 nm ( 7,j)). 

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