. 2018 Apr 25;17:236–245. doi: 10.1016/j.redox.2018.04.017

Table 2.

RBH assay interference tests.

Heme proteins	carbonyls nmoles mg⁻¹ protein
Cyt.c_untr^a	1.15 ± 0.09 (1.09 ± 0.15)^b
Cyt.c_red	0.05 ± 0.01^c (0)^b
Hb_untr^a	5.15 ± 0.07 (5.30 ± 0.15)^b
Hb_red	0.08 ± 0.03^b (0)^b
DNA: its non-specific binding with RBH^a
Tested DNA quantity: in mg and in corresponding {nmoles carbonyls}^b
0.1 mg	0.2 mg
{306}^b	{612}^b
(0 or 0%)^a,c	(0 or 0%)^a,c
[0.09 or 0.03%]^d	[0.18 or 0.03%]^d

Notes on heme protein interference.

^aCarbonyl content (nmoles mg⁻¹ protein) of cyt.c_untr (MW 12,000) and Hb_untr (MW 64,500), compared to their heme content (83 and 62 nmoles, respectively) mg⁻¹, is 80- and 10-fold lower, respectively, which suggests no heme interference on the RBH assay.

^bValues in parentheses, determined by the ntrDNPH assay, have been previously reported [1].

^cCarbonyl values for cyt.c_red and Hb_red obtained by the RBH assay are indicative to its very high detection limit (Table 1).

Notes on DNA interference.

^aDNA % interference is defined as the number of nmoles DNA carbonyl groups (shown by the 1st number in parentheses) that are detected by the RBH assay out of theoretical 100 nmoles contained in the tested quantity of DNA. This is expressed as %, and is shown as such by the 2nd number in parentheses.

^bValues are nmoles DNA carbonyls contained in the tested DNA (0.1 and 0.2 mg), and are derived from the correspondence of 1 µg DNA to 3.06 nmoles carbonyls as shown elsewhere [1].

^cZero values in parentheses (designating same quantities as those in parentheses explained in table's Note ‘a′) are derived from the fluorescence values of the RBH assay's solubilization solution due to the presence of solubilized DNA (after the initial solubilization of its pellet in NaOH). The obtained zero values of carbonyls are explained by the observation that the RBH assay's pH-5-adjusted solubilization solution does not solubilize the DNA that precipitates during the application of the assay (see article's Part C. Standardization of the RBH assay against possible interfering factors). Therefore, the zero value can be due either to the non-solubilization of DNA or to the overrun of the sensitivity limit of the assay to detect the RBH reagent that may be non-specifically bound on the minor quantity of DNA that may have been solubilized. DNA insolubility in the solubilization solution of the RBH assay is the main mechanism by which this assay does not exhibit RBH-DNA interference (even at its minor degree determined 0.03%) when testing carbonyls in protein samples that may be contaminated with DNA.

^dValues in brackets (designating same quantities as those in parentheses explained in table's Note ‘a′) are derived from the fluorescence values of the RBH assay's pH-5-adjusted solubilization solution, measured after initially subjecting the insoluble DNA precipitate to NaOH solubilization (as also described in the preceding table Note ‘c′), and then mixing the resulting solubilizate with the corresponding assay solubilization solutions. The DNA pellet alkaline pre-solubilization procedure is employed in order to determine the degree (0.03%) of the non-specific interference of DNA on the RBH assay.