Lowry method
The Lowry, or increased alkaline copper method that starts with the addition of an alkaline solution of Cu2 + on the test. Copper is a complex with the nitrogen atom in peptide bonds of proteins under this condition, and reduced to Cu +. Cu + which, together with the R-group of tyrosine, tryptophan, and cysteine residues of the protein will be added to react with the Folin-Ciocalteau reagent, the sodium tungstate, sodium molybdate, phosphoric acid, and HCl (W6 + / + MO6). During this reaction, the Cu + is oxidized to Cu3 +, which then reacts with the peptide backbone to the imino peptide (R1-CO-N = C (R2)-Co-R3) and Cu2 +, and Folin reagent is reduced to tungsten molybdenum-blue. Absorption in glass or polystyrene cuvettes at 720 nm, or, if this value is too high (> 2), at 500 Nm.
A calibration curve with standard protein solutions [eg, bovine serum albumin (BSA)] used in the whole protein is not known. Under optimal conditions, and the absence of reactive side chain, have shown that two electrons are transferred per unit of Tetra peptide, however, with significant protein proline or hydroxyproline content, or with the side chain, in the complex of copper (as glutamate) produce less color . Side chain of cysteine, tyrosine and tryptophan to four and four electrons, produce protein respectively.3 Note the different intensity of color is different, especially as a result of a variety of tyrosine and tryptophan content.
With the reagents in the advance, which requires testing Lowry ~ 1 h. A 400 ml sample is required, with 2 to 100 mg of protein (5-250 mg / ml). Non-linear calibration curves obtained from the decomposition Folin reagent for alkaline pH following addition to the sample produced in the reaction is not complete. These agents is that acidify the solution, chelated copper, or a decline in copper (II).
