Protein, copper test

Evid .nce in Support of the Peptide Theory.—(i.) All proteins give a violet or rose colour with copper salts in alkaline solution (the copper protein test), which is characteristic of the peptide linkage =CH —CO—NH—CH=, and not given by any individual amino acid other than serine, threonine and histidine, which have a somewhat similar arrangement in their molecules. 

Apply the copper-protein test to 20 ml. of the mixture. If negative, no unhydrolysed protein is present. 

Copper-protein test followed by acidification. This distin-... 

Copper. In the presence of sulfur dioxide, copper-protein cloudiness may develop in white wines. Only small amounts of copper (about 0.3 to 0.5 mg/liter) cause cloudiness. Widespread use of stainless steel in modern wineries has reduced copper pickup, but many wineries routinely test their wines for copper. Atomic absorption spectrophotometry is the method of choice (51) although reducing sugars and ethanol interfere, and correction tables must be used (107). To reduce this interference, chelating and extracting with ketone is recommended (108). Lacking this equipment colorimetric procedures can be used, especially with di-ethyldithiocarbamate (3, 4, 6, 9,10, 22,109). Neutron activation analysis has been used for determining copper in musts (110). 

According to the induced-rack and the entatic state hypotheses [10,12], the Cu(II) coordination sphere in the blue copper proteins is strained into a Cu(I)-like structure. Such hypotheses are hard to test experimentally, but with theoretical methods it is quite straightforward. The actual coordination preferences of the copper ion can be determined by optimising the geometry of the ion and its ligands in vacuum if the optimised structure is almost the same as in the proteins, strain is probably of minor importance for the geometry. 

This formula is eoneeptually simple, and its application can be facilitated, in the ease of Type 1 copper proteins, by referral to x-ray crystallographic structures. One therefore might test this and similar models by comparing the experimentally measured NQI parameters with estimates derived from the known x-ray structures and routine computational chemistry procedures. 

In alkaline solution biuret, HN(CONH2)2 reacts with copper(II) sulfate to give a characteristic violet colour due to the formation of the complexes [Cu2(/l-OH)2(NHCONHCONH)4] (Fig. 28.6a) and [Cu(NHCONHCONH)2] . This is the basis of the biuret test in which an excess of NaOH solution is added to the unknown material together with a little CUSO4 soln a violet colour indicates the presence of a protein or other compound containing a peptide linkage. 

Prepare 30-fold and 50-fold dilutions of the test solution. Add 1.25 ml of a mixture prepared the same day by combining 2.0 ml of a 20 g/1 solution of copper sulphate R in water R, 2.0 ml of a 40g/l solution of sodium tartrate R in water R and 96.0 ml of a 40 g/1 solution of sodium carbonate R in 0.2M sodium hydroxide to test tubes containing 1.5ml of water R (blank), 1.5ml of the different dilutions of the test solution or 1.5 ml of the reference solutions. Mix after each addition. After approximately 10 min, add to each test-tube 0.25 ml of a mixture of equal volumes of water R and phosphomolybdotungstic reagent R. Mix each addition. After approximately 30 min, measure the absorbance (2.2.25) of each solution at 750 nm using blank as the compensation liquid. Draw a calibration curve (from the absorbances of the eight reference solutions the corresponding protein contents and read from the curve the content of protein in the test solution. 

This method should be preferred if protein concentration has to be determined in the presence of detergents. But if copper chelators, such as EDTA, or reductants, such as 2-mercaproethanol or DTE/DTT or reducing carbohydrates (e.g. > 10 mM glucose), are components of the sample, the test does not work reliably. 

Commercial wines are commonly tested for protein stability. Wine proteins, upon denaturation by heat or cold, may cause cloudiness and unsightly deposits after bottling. In addition, proteins may combine with iron and copper salts to form flocculate material in bottled wines. The reaction and absorption of proteins on bentonite is an effective means of removing protein from wines (109, 110, 111). Therefore, fining wines... 

If you are trying to show only the presence of a protein, a quick test to carry out is known as the Biuret test. A mixture of dilute sodium hydroxide and 1% copper( ii ) sulfate solution is shaken with a sample of the material under test. If a protein is present, a purple colour appears after about three minutes (Figure 15.25). 

Biuret test for proteins. Warm the sample in water, add concentrated sodium hydroxide solution and a drop of very dilute copper(u) sulfate solution. 

The biuret test. This is one of the most general tests for proteins. When a protein reacts with copper(II) sulfate, a positive test is the formation of a copper complex which has a violet color. 

The iron complexes of both human serum transferrin and chicken ovo-transferrin were completely resistant to all proteolytic enzymes tested (7). The metal-free proteins, on the other hand, were rapidly hydrolyzed under similar conditions. Fig. 14 compares results with bovine a-chymo-trypsin. Other enzymes used with similar results were bovine trypsin, bacterial proteinase (Nagarse) and ficin (735). The weaker copper complex of chicken ovotransferrin was hydrolyzed, however, at a rate approx-... 

---