The Nitric Acid Method for Protein Estimation in Biological Samples

|Protein1 Comment
A D V E R T I S E M E N T



In order to quantitate protein in biological samples, these several methods, Bradford, Lowry, BCA, Bohlen, has limitation since the constituents of biological buffers sometimes interfere with those methods. Therefore, it is limiting their application. But don’t worry, there is still a method that could be used to estimate protein in biological samples. The method is Nitric Acid method which is based on the formation of substances, as the result of a reaction between nitric acid and protein, that can be measured by using spectrophotometer at 358 nm.

Materials
Reagents/Equipment

  1. Nitric acid.
  2. BSA, bovine insulin, human fibronectin, human fibrinogen, rat albumin, chicken ovalbumin.
  3. Tris.
  4. Triton X-100, Tween 20, Brij 97, n-octyl-glucoside, digitonin, glycerol, 3-nitrotyrosine, and various amino acids.
  5. Urea (CO(NH2)2).
  6. Electrophoresis grade SDS and beta-mercaptoethanol.
  7. Diode array UV-Vis Spectrophotometer.

Nitric Acid Protein Determination Method

  1. SDS sample buffer: 2% (w/v) SDS; 4 M urea (prepared as an 8 M stock and deionized over Bio-Rad AG501-X8 mixed bed resin prior to use); 62.5 mM Tris-HCl, pH 6.8 at 20-22oC, and 1 mM EDTA with or without 5% (v/v) beta-mercaptoethanol.
  2. ACS reagent grade concentrated (70%) nitric acid.
  3. Bovine Serum Albumin.
Method
Nitric acid Protein Determination Method

The following is the recommended procedure for determining protein content in samples solubilized in SDS sample buffer:

Prior to assay for protein, cells must be washed in serum-free medium or physiological buffer to remove (exogenous) protein and cross-reacting substances contained in culture media.
In contrast to existing assays, the nitric acid method is not adversely affected by urea, reducing agents, and most nonionic and ionic detergents. As a consequence, it is possible to solubilize samples directly in SDS sample buffer, remove an aliquot for protein determination, and then adjust the sample volume based on the result of this assay. One advantage of the nitric acid method, therefore, is that protein content can be assayed on samples solublized in SDS-PAGE sample buffer immediately prior to application of the samples to electrophoresis gels.
  • Remove an aliquot containing 5–100 microgram of protein. Bring sample volume to 10 microliters.
  • Add 140 microliters of 70% nitric acid.
  • Incubate at 20–22oC for 2 h.
    The assay of protein content in detergent-solubilized cells by the nitric acid method requires an incubation period of only 2 h at 22oC. In contrast, similar determinations on nitric acid-solubilized cells require incubation periods of at least 24 h for optimal detection of differences between different cell lines. Longer incubations may be required for nitric acid-solubilized cells because longer times may be required to complete protein nitration under these conditions.
  • Utilizing suitable microcuvettes, measure absorbance at 358 nm with H2O as a blank.
    Water, rather than 70% nitric acid, should be used for a “blank” (or in the reference cell) for all spectrophotometric determinations.
  • Compare results to a standard curve constructed with varying amounts of BSA in 10 microliters SDS sample buffer subjected to the same conditions.
    Proteins with differing tyrosine/amino acid content would be expected to yield different slopes when utilized as standards for this assay. Although this can present a problem in experiments that require the precise quantitation of a single polypeptide species, it does not appear to be a limitation when this method is used to compare relative protein content in complex biological mixtures. Moreover, when the nitric acid assay was applied to a variety of polypeptides (including BSA, rat albumin, chicken ovalbumin, bovine insulin, human fibrinogen, and human fibronectin), the resulting slopes did not correlate with either tyrosine or tryptophan content of the polypeptides, suggesting that tertiary structure or other factors might also influence the extent of nitration during nitric acid treatment. Similar differences in reactivity of various purified polypeptides have been previously observed with other protein estimation methods as well.
    The nitric acid method has a limit of sensitivity of approx 5 micrograms when performed using 100 microliters cuvettes. This sensitivity is slightly lower than the methods of Lowry et al., Smith et al. (BCA) and Bradford performed under similar semimicro conditions.

Nitric Acid Protein Determination Method Modification One: Whole Cells

Simple modifications allowed this procedure to be applied to other settings. When protein content in whole cells is to be determined:

  • Wash cells with serum-free buffer.
  • Solublize cells directly in 70% nitric acid.
  • Incubate at 22oC for 24 h.
  • Determine absorbance at 358 nm (dilution in 70% nitric acid may be necessary).
  • Compare results to a standard curve constructed with varying amounts of BSA subjected to the same assay conditions.

Nitric Acid Protein Determination Method Modification Two: Perchloric or Trichloric Acid Precipitates

When perchloric acid or trichloric acetic acid precipitates are assayed for protein content:

  • Solubilize precipitates directly in nitric acid.
  • Incubate for 2 h at 22oC.
  • Determine absorbance at 358 nm (dilution in 70% nitric acid may be necessary).
  • Compare results to a standard curve constructed with varying amounts of BSA subjected to the same assay conditions.

Hopefully by applying this Nitric Acid method you can determine the Protein concentration of your biological samples.

One Response to The Nitric Acid Method for Protein Estimation in Biological Samples