[Bidagdha_TADIR]

I am not sure whether this should be placed in biochemistry subforum or this subforum, so here I go.

I searched on the internet and went to google books to understand the reactions in Lowry protein assay method. The first reaction is Biuret reaction. Some references are saying that in this reaction the copper ions (in cupric form) will form complex with 4-6 peptide bond nitrogens and cupric ion is converted to cuprous form. Other references are saying the complex is between cupric ions and peptide bond nitrogens. So which is true, in the complex is the copper cupric or cuprous?

Secondly, how melamine gives positive test in Lowry method? My teacher told me that because melamine gives positive test it was added in dairy product in China. At that time I believed him, but now it seems that NH-CO bond is necessary for the assay which melamine doesn't have. So can melamine give positive test to Lowry method?

[Furanone]

The more standard test for Melamine based on AOAC Official Methods is based on Nitrogen Determination multiplied by a conversion factor commonly 6.25 (unless one more specific for that type of food ie 6.38 for milk, 5.75 for flour). Since Proteins are the only macronutrient in food to contain nitrogen (micronutrients include vitamins, phospholipids, etc but negligible compared to proteins) using Kjehldahl acid digestion and titration or more commonly now the Dumas combustion method can accurately give the nitrogen content and thus the % protein when applying the conversion factor.

If you look at the structure of melamine, there are 6 nitrogen atoms along with 3 carbon and 6 hydrogen. By mass though it has 67% nitrogen so by adding a very small amount of melamine, it can make it look like there is a much higher amount of protein in the food. Melamine was forming crystal complexes with cyanuric acid that were giving health problems to animals and children in China from pet food and baby food with melamine added, more specifically crystallization in the kidneys.

As for Lowry method, I thought the chromophore had to do with the phenalalanine, tyrosine and tryptophan while the Biuret as you said had to do with creating a chromophore from the peptide bonds, and I believe the Lowry has a 10-fold sensitivity increase over the Biuret method.

[Arkcon]

Adding to what Furanone: said, melamine doesn't react with the Lowery method.   It reacts with the Kjehldahl nitrogen method, and looks like it has more nitrogen the the similar mass of protein.

[marquis]

Did a lot of work on water soluble proteins for medical.  Used several
methods, including lowry, modified lowry, Bradford, and LEAP.
There were interferences on all of them.  We were always working
with people trying to figure out what was going on.

Arkon mentioned kjeldahl. He's right, it is often used in industry.  But it can
be time intensive. 

The agriculture industry has done a lot of work with near infra-red(NIR) testing for
nitrogen determination.  And, you can go with GC-MS or GC with a
nitrogen phosphorus sulfur detector.

Hope this helps.

[Bidagdha_TADIR]

Okay, so melamine is not detected as protein in Lowry method. That clears up one issue, thank you.

What I understand so far about the Lowry method is this - the copper ions form a complex with 4-6 peptide bond nitrogens. This is the Biuret reaction. Then an electron is transferred from the complex to the Folin-Ciocalteau reagent (i.e. phosphomolybdic acid-phosphotungstinic acid complex).  That electron reduces the Folin-Ciocalteau reagent and causes blue color. Additionally cysteine, tryptophan and specially tyrosine can act as reducing agent (due to side chain or something, I am not sure) and can cause blue color formation independently.

Now this explanation would fit if copper ion in the peptide-copper complex was in the cuprous form. Then the cuprous ion could give an electron to the Folin-Ciocalteau reagent and become cupric ion. So the question is whether the complex formed in Biuret reaction contain cupric ion or cuprous ion.

Since the reagent used for Biuret reaction is basically the Fehling reagent, I am considering that before the Biuret reaction takes place there is cupric ion-tartrate complex. Then the peptide bonds will react with the cupric ion. So, is the complex formed cuprous-peptide complex or cupric-peptide complex? (sorry for the repeat)

Of course, some internet references are saying that Tyr, Trp, Cys are the ones responsible for blue color in Lowry method. Others are saying that the copper-peptide complex is essential for blue color formation. I believe the latter because otherwise there is no need to use the Biuret reaction, we could do this just by Folin-Ciocalteau reagent.

Summery: The reactions taking place in Lowry protein assay method are poorly understood (by me).