General Biology Section A

Bio Lab Report – Biochemistry

Group 9:
Sergio Nicholas Arifin (2020220044)
Ellena Brilian (2019120008)
Maria Tarmidi (2020920001)
Proceeding the study of biochemistry, this paper is intended to be an introduction of
sorts to the ever-present compound in all living beings: protein, an essential and
significant macronutrient which functions as a source of energy that in turn, supports
cellular growth.
1. Biuret assay’s ability to give a positive reaction with amino acids
Biuret assay or Piotrowski test is a biochemical assay that lets people
accurately quantify protein concentration in a material within the range of 5 –
150 mg/ml. An assay itself is a process of analysing a substance to determine
its composition or quality—the term frequently used in the mining industry to
refer to tests of ore or minerals while a biuret is a small compound that forms
when urea is heated which causes two urea molecules to join. Said reagent can
be obtained by mixing in copper (II) sulphate pentahydrate with sodium
potassium tartrate, sodium hydroxide, and potassium iodide in deionized or
distilled water. Urea molecules fused in this manner produce amide groups (-
NH) at the centre of the molecule which bind to copper ions at basic pH. The
copper complexes that result from this interaction produce a strong blue colour
that can be measured with a spectrophotometer.

Proteins also contain amide groups. When an amino group and a carboxyl
group join to form a peptide bond, the amino group (-NH2) becomes an amide
group (-NH). Therefore, proteins will also complex with copper ions at a basic
pH then the copper ions will complex with the amide groups in the proteins to
create a blue colour that can be measured using the same aforementioned
device. The more blue there is in a sample, the more quantity of protein it
contains. These protein-packed samples would be constituted of polypeptides
which are made of amino acids that are conjoined with peptide bonds. The
longer the polypeptide chain is within a substance, the darker the hue of the
biuret gets. The shorter it is, the paler the hue gets.

The biuret assay cross-reacted with several amino acids, dipeptides, and other
organic compounds able to form 5- or 6-member ring chelation complexes with
copper. Reactions with amino acids and dipeptides had higher absorbance
maxima (blue color) than with larger peptides and proteins (purple).
Compounds forming potential 4-, 7-, 8-, or 9-member ring complexes with
copper had low reactivity. Amino acid amides, dipeptides, and longer peptides
had substantial reactivity, except those containing proline.

2. Other tests capable of detecting the presence of protein

- Ninhydrin test
It is another color test for proteins that indicates the presence of amino acids in
a solution. Only alpha amino acids give a positive Ninhydrin test. Ninhydrin is
 a powerful oxidizing agent that causes oxidative decarboxylation of the alpha
amino acids forming an aldehyde, ammonia, and Hydrindantin (reduced form
of Ninhydrin). This Hydrindantin reacts with ammonia and another Ninhydrin
molecule to form a bluish-purple colored complex.

- Xanthoproteic Test

Xanthoproteic test is used to detect amino acids containing an aromatic

nucleus (tyrosine, tryptophan and phenylalanine) in a protein solution which
gives yellow color nitro derivatives on heating with conc. HNO3. The aromatic
benzene ring undergoes nitration to give yellow colored product. Phenylalanine
gives negative or weakly positive reaction though this amino acid contains
aromatic nucleus because it is difficult to nitrate under normal condition. On
adding alkali to these nitro derivative salts, the color changes from yellow to
orange. This test is used to differentiate aromatic amino acids which give
positive result from other amino acid.

- Millon’s Test
Compounds containing hydroxybenzene radical react with Millon’s reagent to
form red complexes. The only amino acid having hydroxybenzene ring is
tyrosine. Thus, this test is specific for the amino acid tyrosine and the protein
containing this amino acid. Tyrosine when reacted with acidified mercuric
sulphate solution gives yellow precipitate of mercury-amino acid complex. On
addition of sodium nitrate solution and heating, the yellow complex of
mercury-amino acid complex converts to mercury phenolate which is in red
color.
- Lead Sulfide Test
The test is based on the principle of detection of sulfur in a solution by the
degradation of the S-H or S-S group in amino acids under strongly alkaline
conditions. Amino acids like cysteine and cystine release sulfur in the presence
of strong alkaline conditions at a high temperature. The sulfur then combines
with the alkali (NaOH) to form Na2 The Na2S thus formed reacts with lead
acetate to form lead sulfide, which results in a black residue. For the reaction to
take place, free sulfur ions should be present in the medium.

- Hopkin’s Cole Test

The test is based on the principle that the layering of concentrated sulfuric acid
over a mixture of tryptophan-containing proteins with the Hopkin’s Cole
reagent results in the formation of a violet ring at the interface. The glyoxylic
acid added to the sample combines two tryptophan molecules by acting on the
indole ring of the tryptophan molecules. The condensation product thus formed
undergoes dehydration to form a violet colored pigment. The color developed
in the test is due to the indole group of the tryptophan molecule, which is
converted into a colored compound by oxidation brought about by the aldehyde
group of glyoxylic acid. The H2SO4 added to the reagent helps to stabilize the
glyoxylic acid and prevent its decomposition and the release of carbon dioxide.

3. Brief explanation of the Covid-19 principle antigen test

COVID-19 Antigen Rapid Test is a rapid membrane-based lateral flow
immunoassay for the qualitative detection of SARS-CoV-2 antigens in human
nasopharyngeal and nasal swab specimens. During the test, any SARS-CoV-2
antigens contained in the sample react with the antibody-coated nanoparticles
contained in the reactive test strip. As a result, the blend migrates
chromatographically by capillary action along the reactive strip.
If the specimen contains SARS-CoV-2 antigens, these will bind to SARS-CoV-
2 antibodies contained in the (T) Test line region and generate a colored line on
the test strip, indicating a positive result. If the antigens are not present in the
specimen, no colored line will appear in the (T) Test line region, indicating a
negative result. As a procedural control, a colored line will always appear in
the (C) Control line region, indicating the test procedure has been performed
properly and that test components have worked as intended.