Spectrometric Evaluation of the Approximate pK of the Carboxyl Group in 2,4-Dinitrophenyl-Amino Acids

Ramachandran, L. K.; Sastry, L. V. S.

doi:10.1021/bi00907a012

Cited by 22 publications

(5 citation statements)

References 7 publications

(2 reference statements)

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“…Peptides were sonicated for 3 minutes in a water bath (~25 °C, Solid State Ultrasonic FS-9, Fisher Scientific, Birmingham, AL), maintained on ice and used in assays within two hours of preparation. The molar concentrations of Abz-KSKTKC(farnesyl)VIK-Dnp and VIK-Dnp were determined by measurement of A 360nm and use of a molar extinction coefficient value of ε = 17,530 M −1 cm −1 for ε-DNP-lysine [28]. The molar concentration of Abz-KSKTKC(farnesyl) was determined by measurement of A 320nm and use of a molar extinction coefficient value of ε = 21,870 M −1 cm −1 for Abz [29].…”

Section: Methodsmentioning

confidence: 99%

Acquisition of accurate data from intramolecular quenched fluorescence protease assays

Arachea

Wiener

2017

Analytical Biochemistry

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The Intramolecular Quenched Fluorescence (IQF) protease assay utilizes peptide substrates containing donor-quencher pairs that flank the scissile bond. Following protease cleavage, the dequenched donor emission of the product is subsequently measured. Inspection of the IQF literature indicates that rigorous treatment of systematic errors in observed fluorescence arising from inner-filter absorbance (IF) and non-specific intermolecular quenching (NSQ) is incompletely performed. As substrate and product concentrations vary during the time-course of enzyme activity, iterative solution of the kinetic rate equations is, generally, required to obtain the proper time-dependent correction to the initial velocity fluorescence data. Here, we demonstrate that, if the IQF assay is performed under conditions where IF and NSQ are approximately constant during the measurement of initial velocity for a given initial substrate concentration, then a simple correction as a function of initial substrate concentration can be derived and utilized to obtain accurate initial velocity data for analysis.

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Section: Methodsmentioning

confidence: 99%

Acquisition of accurate data from intramolecular quenched fluorescence protease assays

Arachea

Wiener

2017

Analytical Biochemistry

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“…Dry weights were corrected for the weight of buffer present. The degree of DNP substitution was estimated spectrally from the absorbancy at 360 m]z assuming all DNP to be present on the epsUon-amino groups of lysine and using the molar extinction coefficient for free N,e-2,4-dinitrophenyl-L-lysine (e-DNP-L-lysine) at 360 m# (17,530) (17). A single preparation of DNP-BGG containing 60 DNP groups/mole and a single preparation of DNP-BF containing 150 groups/mole were used throughout.…”

Section: Methodsmentioning

confidence: 99%

Studies on the Control of Antibody Synthesis

Siskind

Dunn

Walker

1968

The Journal of Experimental Medicine

112

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The effect of antigen dose on the kinetics of circulating antibody synthesis and on antibody affinity was studied in a haptenic system. High doses of antigen resulted, early in immunization, in higher concentrations of antibody followed later in the immune response by decreased serum levels of antibody as compared with lower doses of antigen. The affinity of the initial antibody synthesized was very similar over a wide antigen dose range. Subsequently, however, a rapid rise in affinity was seen in animals immunized with low doses of antigen, while relatively little change in affinity was seen in animals immunized with higher antigen doses. Suppression of active antibody formation by passive antiserum led to an increase in antibody affinity. The results are discussed in terms of the mechanisms involved in the selection of a population of cells to participate in the immune response and the mechanisms whereby antigen dose and circulating antibody function to control antibody synthesis.

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“…The "dry weights" so obtained were corrected for the weight of buffer present. The degree of DNP substitution was estimated spectraUy by assuming that all hapten groups were coupled to C-amino groups of lysine and by using the molar absorbancy of free e-DNP-L-lysine [17,530 at 360 m# (12)]. The degree of substitution of R-Azo derivatives was very roughly estimated (13) from their absorbancy in alkali at 500 mtz, assuming all R-Azo groups were coupled to tyrosine residues.…”

Section: Methodsmentioning

confidence: 99%

Studies on the Control of Antibody Synthesis

Brody

Walker

Siskind

1967

The Journal of Experimental Medicine

105

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In the system studied, antigenic competition between two haptenic determinants was found to be of the same extent whether the haptens were on the same or on separate carrier molecules. Suppression of antibody formation to one determinant by administration of passive antibody partially eliminated the depressive effects of antigenic competition when the two haptens were located on separate carrier molecules but had no effect on antibody production to the second hapten when the two determinants were present on the same molecule. The results are discussed in terms of the mechanisms of suppression, antigenic competition, and control of antibody formation.

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Spectrometric Evaluation of the Approximate pK of the Carboxyl Group in 2,4-Dinitrophenyl-Amino Acids

Cited by 22 publications

References 7 publications

Acquisition of accurate data from intramolecular quenched fluorescence protease assays

Acquisition of accurate data from intramolecular quenched fluorescence protease assays

Studies on the Control of Antibody Synthesis

Studies on the Control of Antibody Synthesis

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