Time-resolved Chemiluminescence Technique for the Microdetermination of Proteins Based on Their Complexation with Copper(II)

Abstract

As the proteomics project has progressed during recent years, scientists are purifying and identifying ever smaller proteins that are active at very low concentrations in biological fluids. The routine methods of protein determination (such as the Lowry 1 and Bradford 2 methods) are not possible to control the protein concentration in these studies due to their low sensitivities. Although a high sensitivity of protein assay can be obtained by silver staining, 3,4 colloidal gold staining 5 and fluorometric methods, 6-9 they require a complicated procedure or expensive reagent. Recently, a resonance light scattering (RLS) technique, as a new spectral method, has been widely studied to determine proteins based on protein aggregation, or the interaction between proteins and dyes. [10][11][12][13][14] However, RLS generally suffers from the disadvantage of an instable response signal resulting from serious effects of the experimental and instrumental conditions. Also, most of the RLS assays can only allow the determination of proteins at concentrations higher than 0.1 µg/ml because of their high background. It is thus still desired to develop some sensitive, inexpensive and rapid techniques for the determination of proteins.It is well known that the chemiluminescence (CL) method can achieve high sensitivity by using a simple instrument. It has been exploited with a wide range of applications in different fields. [15][16][17] However, the direct detection of proteins with the CL method has been restricted because the general proteins can not produce CL signal directly. Hara et al. have described a series of methods for protein determination based on the suppression of CL reactions catalyzed by metal ions. [18][19][20] In previous papers, 21,22 it has been demonstrated that an unsaturated complex of Cu(II) with proteins has a strongly catalytic effect on the luminol and 1,10-phenanthroline CL reaction. Based on this result, a simple and sensitive CL assay for proteins has been established. However, all of these CL methods for protein determination were based on the difference in the CL intensity between metal ions and the complexes of metal ions with proteins. Thus, these CL methods must be applied under rigid experimental conditions, and the sensitivity has been limited by the high CL background.In the present work, we discovered that CL emission produced by an unsaturated complex of Cu(II) with proteins is much more long-lived than that by free Cu(II). The CL signals originated from proteins were identified and measured with a timeresolved technique. Due to the decrease in the CL background by using a time-resolved technique, the proposed method has about an order of magnitude higher sensitivity for protein detection compared to that of the former CL method. Experimental ApparatusThe CL intensity and intensity-time profiles were measured and recorded with a BPCL ultra-weak luminescence analyzer and software BPCL172 (Institute of Biophysics Academia Sinica, Beijing, China Based on the complexation between proteins and...

Spectroscopic and molecular modeling studies of the interaction between cytidine and human serum albumin and its analytical application

“…The most frequently used methods are Biuret, 8 Bromocresol Green, 9 Coomassie Brilliant Blue, 10 and Bromophenol Blue. 11 In recent years, novel methods such as spectrophotometry, 12 spectrofluorometry, 13 Resonance Light Scattering (RLS), 14 and chemiluminescence 15 have been developed. The most quantitative analysis for proteins is generally based on their fluorescence enhancement effect on organic dyes.…”

Protein analysis with terbium (III) and sodium dodecyl sulphonate by a second‐order scattering technique

“…Quantitative analysis of proteins is very important and detection methods include spectrophotometry (1) and spectrofluorimetry (2) and the commonly used Lowry (3), Bradford (4) and bromophenol blue (5) methods. In addition, chemiluminescence (6), electrochemiluminescence (7) and resonance light scattering (RLS) methods for the determination of proteins have been reported (8)(9)(10).…”