| Protein is one of the most important materials in organism, and a foundation of the life. The quantitative analysis of protein is a basic requisite not only in biochemistry, pharmacy and other biological subjects, but also in clinical assay and food survey, because protein levels are often used as a reference for measurements of other components in biological systems. So it is of great significance to establish rapid, accurate and sensitive protein assay methods. In this paper, five highly sensitive new methods for the determination of protein were developed by studying on the binding reactions of arsenazo (carboxyazo)-metal ion complexes with protein and have been applied to the analysis of total protein in biological samples with satisfactory results. The main research contents are as follows: 1. A new method for the determination of proteins by using arsenazo Ⅲ-yttrium( Ⅲ) complex as a spectral probe was established. This method is based on the binding reaction of protein with arsenazo Ⅲ-yttrium(Ⅲ) complex in Briton-Robinson buffer at pH 2.32.5. The binding reaction is complete within 30 min at room temperature, and causes absorbance decrease at 652 nm of the complex. The decrease of absorbance is proportional to the concentration of protein. The calibration curve for human serum albumin (HSA) is linear up to 20 mg/L, and the apparent molar absorptivity of binding reaction at 652 nm is 2.6×106 L·mol-1·cm-1. The method has been applied to the determination of the total protein in the human serum samples with satisfactory results. 2. The binding reaction between protein and p-acetylarsenazo-yttrium(Ⅲ) complex was studied and the optimum conditions of reaction were chosen by spectrophotometry. Based on the binding reaction, a novel method was established for the spectrophotometric determination of protein by using p-acetylarsenazo-yttrium( Ⅲ) complex as a spectral probe. The binding reaction is complete in HAc-NaAc buffer at pH 3.7-4.0, and causes absorbance decrease at 653 nm of the complex. The decrease of absorbance is proportional to the concentration of protein in the range of 0-20 mg/L for HSA and the apparent molar absorptivity of binding reaction at 653 nm is 1.98×106 L·mol-1·cm-1 for HSA. The method has been applied to the determination of the total protein in the human serum samples with satisfactory results. 3. This is the first report on the binding reaction between βtype complex and protein. In HCl-NaAc buffer medium at pH 0.75-1.5, protein binds nitrocarboxyazo-barium( Ⅱ) βtype complex rapidly (within 7 min at room temperature) to form a product stable for 1.0 h, leading to the change of the absorption spectrum of the complex and the decrease in the absorbance at 710 nm. Based on this, a sensitive method for the determination micro amounts of protein was proposed. The calibration curve for HSA is linear in the range of 020 mg/L with a detection limit of 0.015 mg/L. Very few coexisting substances interfere with the assay. The method has been applied to determinate the total protein in human urine samples with the same results as by the Bradford method. 4. Based on the strong absorbance of p-acetylcarboxylazo-strontium(Ⅱ) βtype complex at 712 nm which can be decreased by the addition of protein in HCl-NaAc buffer at pH 0.81.0, a novel quantitative method was developed for the determination of protein. Under the optimum conditions, the linear ranges of calibration curves for the determination of HSA, bovine serum albumin (BSA), egg albumin (OVA) and human immunoglobulin (HIG) were 010 mg/L, 010 mg/L, 025 mg/L and 020 mg/L, respectively. The detection limits were 0.017 mg/L for HSA, 0.018 mg/L for BSA, 0.048 mg/L for OVA and 0.025 mg/L for HIG. The method has been applied to the analysis of total protein in humanurine samples and the results were in accordance with those obtained by the Bradford method, which indicates that the present method is not only sensitive, simple, but also reliable and suitable for practical applications. 5. The binding reaction between m-carboxyllcarboxylazo-strontium( Ⅱ) βtype complex and protein was studied. Under the optimum conditions, four proteins, including HSA, BSA, OVA and HIG were tested. The apparent molar absorptivities of the binding reaction at 704 nm are 5.06×106 L·mol-1·cm-1 for HSA, 4.17×106 L·mol-1·cm-1 for BSA, 1.14×106 L·mol-1·cm-1 for OVA, 8.04×106 L·mol-1·cm-1 for HIG and the linear ranges of calibration curves are 010 mg/L, 010 mg/L, 020 mg/L and 015 mg/L, respectively. The method has been applied to determinate the total protein in human serum samples with the same results as by the Bradford method. Moreover, the mechanism of the binding reaction was explored primarily. It was considered that the electrostatic force was the main binding force. |
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