The primary role of copper-containing proteins is redox function. Most of the currently known copper-containing enzymes react directly with molecular oxygen. Approximately 95% of copper in plasma is bound to oxidase enzymes with α-2-globulin, ceruloplasmin, and oxidase enzymes with ferroxidase activity. Diseases associated with copper deficiency include heart disease, osteoporosis, osteoarthritis, Menkes syndrome, and Wilson's disease. Also, decreased antioxidant function in the body has also been reported. On the other hand, excess copper can be toxic.

This method determines copper concentration by observing the color change in the visible region by the formation of a chelate complex between 3-5 DiBr-PAESA and copper.

Copper bound to ceruloplasmin and proteins is dissociated with a weak acid or denaturant to form a copper-3-5 DiBr-PAESA complex. The copper concentration can be determined by measuring this copper chelate complex at a wavelength of 580 nm.

◯ Samples that can be directly quantified with this kit

Examples: serum, plasma

The denaturant in the kit dissociates copper from the protein and develops color after reducing agent reduces it. No specific pretreatment is required.

※ Do not use blood collection tubes containing EDTA which may affect the assay value.

◯ Samples that require acid extraction

Examples: cell lysates, tissue homogenates, and other aqueous solution samples

The pH of the samples was adjusted to low level to dissociates the copper from the protein, and the supernatant was used as the assay sample. However, only the following chemicals can be measured.

・Free copper

・Protein-bound (coordinated) copper (Protein-Cu2+, e.g. albumin, metallothionein)

・Other coordination-bound copper

※ Do not use lysis buffers containing EDTA as they may affect the measured values.

※ Acid digestion should be performed in the case of organic copper and heme-copper.

◯ Samples that require microwave method and organic matter mixed acid decomposition

Example: Samples containing strong chelating agents such as EDTA, organic zinc (-C-Cu-C- single bonded), copper encapsulated in a cyclic ligand (Cu-porphyrin, etc.)

Acid extraction is necessary when measuring chemical species that cannot be dissociated or samples containing interfering substances such as strong copper chelating agents.

※ After decomposition of organic matter, adjust the pH to 2-3, and use the sample as the assay sample.

(1) Add 12 µl of DIW, standard solution, and assay sample to wells of a 96-well plate.

(2) Add 240 µl of chromogenic solution to well

(3) Incubate the plate at room temperature for 10 minutes.

(4) Measure absorbance at main wavelength 580 nm.

(5) Calculate the copper concentration using the following formula:

(1) Measurement of copper concentration in control serums using this kit

Figure 1: Results of measurement of control serum with known copper concentration.

(2) Measurement of copper concentration in pig liver using this kit

Figure 2: Results of pig liver copper concentration measurement using the ICP-OES method and this kit

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