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Why do protein solutions have to be alkalised in biuret test?

Rosetta ab initio prediction and protein-protein interaction fitness helpNaCl role in CTAB - DNA complex in DNA extractionAre There Rules for How Proteins Are Formed?How do I identify the protein with the highest Disulfide bond density? i.e protein with highest ratio of Disulphide bonds per Peptide bond?Amino acid compatibilityMultiple transcripts encoding for one proteinHow much nucleoside triphosphate is required to form one peptide bond during protein synthesis?How would one identify cellular transcription factors associated with a viral protein in a treated cell line?Is the signal peptide always cleaved during protein synthesis in the ER?How to calculate the amount of protein synthesized knowing the amount of essential amino acids ingested?

5

$begingroup$

I’ve read that CuSO₄ solution reacts with peptide bonds that connect amino acids to create a violet colour, but the instructions always tell me to add NaOH solution to the protein solution before I add CuSO₄. How is alkalising the protein solution before adding CuSO₄ solution an aid to this process?

proteins lab-techniques food-chemistry

share|improve this question

edited May 10 at 5:49

WYSIWYG

31.6k750134

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

$endgroup$

• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  The directions explicitly state to add the sodium hydroxide before the copper sulfate? I've never done the biuret test myself, but I can't imagine it would make a difference. Commercially available biuret reagents are sold as premixed solutions.
  $endgroup$
  – canadianer
  May 9 at 22:54
  

  
  
  
  


• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  Can you link the recipe you are talking about?
  $endgroup$
  – Chris♦
  May 10 at 7:04
  

  
  
  
  

add a comment | 

5

$begingroup$

I’ve read that CuSO₄ solution reacts with peptide bonds that connect amino acids to create a violet colour, but the instructions always tell me to add NaOH solution to the protein solution before I add CuSO₄. How is alkalising the protein solution before adding CuSO₄ solution an aid to this process?

proteins lab-techniques food-chemistry

share|improve this question

edited May 10 at 5:49

WYSIWYG

31.6k750134

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

$endgroup$

• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  The directions explicitly state to add the sodium hydroxide before the copper sulfate? I've never done the biuret test myself, but I can't imagine it would make a difference. Commercially available biuret reagents are sold as premixed solutions.
  $endgroup$
  – canadianer
  May 9 at 22:54
  

  
  
  
  


• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  Can you link the recipe you are talking about?
  $endgroup$
  – Chris♦
  May 10 at 7:04
  

  
  
  
  

add a comment | 

$begingroup$

I’ve read that CuSO₄ solution reacts with peptide bonds that connect amino acids to create a violet colour, but the instructions always tell me to add NaOH solution to the protein solution before I add CuSO₄. How is alkalising the protein solution before adding CuSO₄ solution an aid to this process?

proteins lab-techniques food-chemistry

share|improve this question

edited May 10 at 5:49

WYSIWYG

31.6k750134

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

$endgroup$

share|improve this question

edited May 10 at 5:49

WYSIWYG

31.6k750134

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

share|improve this question

edited May 10 at 5:49

WYSIWYG

31.6k750134

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

edited May 10 at 5:49

WYSIWYG

31.6k750134

edited May 10 at 5:49

WYSIWYG

31.6k750134

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

asked May 9 at 22:35

Ubaid HassanUbaid Hassan

1314

2 Answers
2

active

oldest

votes

4

$begingroup$

This is probably to prevent precipitation of copper hydroxide (see Itzhaki & Gill, 1964 - they suggest adding dilute copper sulfate slowly to the NaOH solution to avoid this). If you have the protein already alkalized and ready to react you'll get the color reaction before precipitate forms.

Commercial premixed solutions, like @canadianer mentioned in a comment, have tartrate present to prevent this (Geiger & Bessman, 1972 mention this).

It doesn't seem like it's strictly necessary to add NaOH first, but that's probably the most reliable way to do the assay without using an additional stabilizing agent.

---

Itzhaki, R. F., & Gill, D. M. (1964). A micro-biuret method for estimating proteins. Analytical biochemistry, 9(4), 401-410.

Geiger, P. J., & Bessman, S. P. (1972). Protein determination by Lowry's method in the presence of sulfhydryl reagents. Analytical biochemistry, 49(2), 467-473.

share|improve this answer

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

$endgroup$

• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  Wouldn’t adding sodium hydroxide solution to copper sulfate solution encourage the formation of copper hydroxide? en.m.wikipedia.org/wiki/Copper(II)_hydroxide
  $endgroup$
  – Ubaid Hassan
  May 10 at 7:24
  

  
  
  
  


• 
  
  
  

  
  

  
  
  
  
  
  

  
  $begingroup$
  @UbaidHassan Yes exactly, which is why the OPs recipe tells them to add the hydroxide solution to the protein sample first. Might be confusing since my first reference also suggested adding copper sulfate slowly to sodium hydroxide, but that's only because the worst approach would be to add copper sulfate quickly or adding hydroxide to a concentrated copper solution. You use the same procedure with, for example calcium containing solutions that tend to form precipitate: precipitate is minimized when you keep the metal concentration low.
  $endgroup$
  – Bryan Krause♦
  May 10 at 13:19
  

  
  
  
  

add a comment | 

1

$begingroup$

You mix those two chemicals to get a Cu(OH) 2 colloid, if you mixed them earlier it would settle down and not form a colorful complex (as efficiently). Base is added first because (i presume) it denaturates the protein which partly unfolds it, allowing copper ions to form complexes along a greater surface, giving clearer results.

share|improve this answer

answered May 9 at 23:33

Francis L.Francis L.

1265

$endgroup$

add a comment | 

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4

$begingroup$

This is probably to prevent precipitation of copper hydroxide (see Itzhaki & Gill, 1964 - they suggest adding dilute copper sulfate slowly to the NaOH solution to avoid this). If you have the protein already alkalized and ready to react you'll get the color reaction before precipitate forms.

Commercial premixed solutions, like @canadianer mentioned in a comment, have tartrate present to prevent this (Geiger & Bessman, 1972 mention this).

It doesn't seem like it's strictly necessary to add NaOH first, but that's probably the most reliable way to do the assay without using an additional stabilizing agent.

---

Itzhaki, R. F., & Gill, D. M. (1964). A micro-biuret method for estimating proteins. Analytical biochemistry, 9(4), 401-410.

Geiger, P. J., & Bessman, S. P. (1972). Protein determination by Lowry's method in the presence of sulfhydryl reagents. Analytical biochemistry, 49(2), 467-473.

share|improve this answer

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

$endgroup$

• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  Wouldn’t adding sodium hydroxide solution to copper sulfate solution encourage the formation of copper hydroxide? en.m.wikipedia.org/wiki/Copper(II)_hydroxide
  $endgroup$
  – Ubaid Hassan
  May 10 at 7:24
  

  
  
  
  


• 
  
  
  

  
  

  
  
  
  
  
  

  
  $begingroup$
  @UbaidHassan Yes exactly, which is why the OPs recipe tells them to add the hydroxide solution to the protein sample first. Might be confusing since my first reference also suggested adding copper sulfate slowly to sodium hydroxide, but that's only because the worst approach would be to add copper sulfate quickly or adding hydroxide to a concentrated copper solution. You use the same procedure with, for example calcium containing solutions that tend to form precipitate: precipitate is minimized when you keep the metal concentration low.
  $endgroup$
  – Bryan Krause♦
  May 10 at 13:19
  

  
  
  
  

add a comment | 

4

$begingroup$

This is probably to prevent precipitation of copper hydroxide (see Itzhaki & Gill, 1964 - they suggest adding dilute copper sulfate slowly to the NaOH solution to avoid this). If you have the protein already alkalized and ready to react you'll get the color reaction before precipitate forms.

Commercial premixed solutions, like @canadianer mentioned in a comment, have tartrate present to prevent this (Geiger & Bessman, 1972 mention this).

It doesn't seem like it's strictly necessary to add NaOH first, but that's probably the most reliable way to do the assay without using an additional stabilizing agent.

---

Itzhaki, R. F., & Gill, D. M. (1964). A micro-biuret method for estimating proteins. Analytical biochemistry, 9(4), 401-410.

Geiger, P. J., & Bessman, S. P. (1972). Protein determination by Lowry's method in the presence of sulfhydryl reagents. Analytical biochemistry, 49(2), 467-473.

share|improve this answer

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

$endgroup$

• 
  
  
  

  
  1
  

  
  
  
  
  
  

  
  $begingroup$
  Wouldn’t adding sodium hydroxide solution to copper sulfate solution encourage the formation of copper hydroxide? en.m.wikipedia.org/wiki/Copper(II)_hydroxide
  $endgroup$
  – Ubaid Hassan
  May 10 at 7:24
  

  
  
  
  


• 
  
  
  

  
  

  
  
  
  
  
  

  
  $begingroup$
  @UbaidHassan Yes exactly, which is why the OPs recipe tells them to add the hydroxide solution to the protein sample first. Might be confusing since my first reference also suggested adding copper sulfate slowly to sodium hydroxide, but that's only because the worst approach would be to add copper sulfate quickly or adding hydroxide to a concentrated copper solution. You use the same procedure with, for example calcium containing solutions that tend to form precipitate: precipitate is minimized when you keep the metal concentration low.
  $endgroup$
  – Bryan Krause♦
  May 10 at 13:19
  

  
  
  
  

add a comment | 

$begingroup$

This is probably to prevent precipitation of copper hydroxide (see Itzhaki & Gill, 1964 - they suggest adding dilute copper sulfate slowly to the NaOH solution to avoid this). If you have the protein already alkalized and ready to react you'll get the color reaction before precipitate forms.

Commercial premixed solutions, like @canadianer mentioned in a comment, have tartrate present to prevent this (Geiger & Bessman, 1972 mention this).

It doesn't seem like it's strictly necessary to add NaOH first, but that's probably the most reliable way to do the assay without using an additional stabilizing agent.

---

Itzhaki, R. F., & Gill, D. M. (1964). A micro-biuret method for estimating proteins. Analytical biochemistry, 9(4), 401-410.

Geiger, P. J., & Bessman, S. P. (1972). Protein determination by Lowry's method in the presence of sulfhydryl reagents. Analytical biochemistry, 49(2), 467-473.

share|improve this answer

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

$endgroup$

share|improve this answer

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

answered May 9 at 23:27

Bryan Krause♦Bryan Krause

20.3k33358

1

$begingroup$

You mix those two chemicals to get a Cu(OH) 2 colloid, if you mixed them earlier it would settle down and not form a colorful complex (as efficiently). Base is added first because (i presume) it denaturates the protein which partly unfolds it, allowing copper ions to form complexes along a greater surface, giving clearer results.

share|improve this answer

answered May 9 at 23:33

Francis L.Francis L.

1265

$endgroup$

add a comment | 

1

$begingroup$

You mix those two chemicals to get a Cu(OH) 2 colloid, if you mixed them earlier it would settle down and not form a colorful complex (as efficiently). Base is added first because (i presume) it denaturates the protein which partly unfolds it, allowing copper ions to form complexes along a greater surface, giving clearer results.

share|improve this answer

answered May 9 at 23:33

Francis L.Francis L.

1265

$endgroup$

add a comment | 

$begingroup$

You mix those two chemicals to get a Cu(OH) 2 colloid, if you mixed them earlier it would settle down and not form a colorful complex (as efficiently). Base is added first because (i presume) it denaturates the protein which partly unfolds it, allowing copper ions to form complexes along a greater surface, giving clearer results.

share|improve this answer

answered May 9 at 23:33

Francis L.Francis L.

1265

$endgroup$

share|improve this answer

answered May 9 at 23:33

Francis L.Francis L.

1265

answered May 9 at 23:33

Francis L.Francis L.

1265

answered May 9 at 23:33

Francis L.Francis L.

1265