The Variable Reagent Blank: Protein Determination as a Model

Abstract

Three total protein assays were analyzed to determine the extent of deviation encountered when a constant measured reagent blank is compared to a continuously decreasing true reagent blank. This blank effect owes its regressive nature to the consumption of the active reagent ingredient by the protein reactive species, variably and sometimes, with certain reactants, nonlinearly in the presence of increasing protein concentrations. However, the blank effect of interest here is apparent only when the reagent and the reagentprotein complex present overlapping spectra and therefore absorb at the wavelength of measurement. Thus it was found that while the biuret and the Coomassie brilliant blue assays produced pronounced differences in the variable true reagent blanks, the Folin-Ciocalteau reaction did not develop a deviation from the true blank since the reagent blank does not absorb to any extent at the assay wavelength. In this manner, the latter procedure could serve äs a marker against which the former two blank reactions can be shown to display relatively excessive deviations. Der unterschiedliche Reagenzienleerwert: Proteinbestimmimg als ModellZusammenfassung: Drei Methoden zur Bestimmung von Gesamtprotein wurden untersucht, um das Ausmaß der Abweichung zu bestimmen, das auftritt, wenn ein gemessener konstanter Reagenzienleerwert mit einem kontinuierlich abfallenden wahren Reagenzienleerwert verglichen wird. Dieser von der Analytkonzentration abhängige Leerwerteffekt beruht auf dem Verbrauch von reaktiven Bestandteilen des Reagenz durch die reaktive Proteinspezies. Er ist in Gegenwart steigender Proteinkonzentrationen variabel und mit bestimmten Reaktionspartnern manchmal nicht-linear. Der hier interessierende Leerwerteffekt ist jedoch nur zu beobachten, wenn Reagenz und Reagenz-Protein-Komplex sich überlappende Absorptionsspektren aufweisen und deshalb beide bei der Meßwellenlänge absorbieren. So wurde gefunden, daß zwar das Biuret-als auch das Coomassie-Brilliant-Blau-Verfahren ausgeprägte Differenzen in den variablen wahren Reagenzienleerwerten verursachten, die Folin-Ciocalteau-Reakuon aber keine Abweichung vom wahren Leerwert aufwies, da hier der Reagenzienleerwert bei der Meßwellenlänge nicht absorbiert. Auf diese Weise kann das Folin-Ciocalteau-Verfahren als Prüfmethode dienen, die relativ erheblichen Abweichungen der Leerwertreaktion der beiden anderen Methoden aufzuzeigen. on of the reagent blank can be a minor consideration In some analytical procedures, the eontribution of (1). However, if the active constituent of the reagent the reagent blank is of a constant nature, Except for blank has a spectrum overlapping that of the circumstances such äs those involving inorganic ion equilibrium reaction product generated and is a conmeasurement in which the analyte of interest may be sumable in creating the latter, the true reagent blank a contaminant pf the water of the blank, subtraction is actually regressive in the final measurement. Thus,

Intracavitary Ligand Distribution in Tear Lipocalin by Site-Directed Tryptophan Fluorescence

“…Purity of mutant proteins was verified by SDS-tricine gel electrophoresis ( 2 ). The protein concentrations of stock solutions were determined by the biuret method ( 24 ), and dilute protein concentrations were determined by the Lowry method. In addition, to avoid any discrepancy, absorbance of each protein solution at 280 nm was measured.…”

Site-Directed Tryptophan Fluorescence Reveals the Solution Structure of Tear Lipocalin: Evidence for Features That Confer Promiscuity in Ligand Binding

“…The purity of mutant proteins was verified by SDS-Tricine gel electrophoresis (1). The protein concentration was determined by the biuret method (19). For ligand binding experiments, TL-WT and mutants were delipidated with chloroform and methanol as described previously (1).…”

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

“…Purity of mutant proteins was verified by SDS tricine gel electrophoresis [23]. The protein concentrations were determined by the biuret method [24].…”