Quantitative tests for proteins - PowerPoint Presentation

Slide1

Quantitative tests for proteins

Introduction

Colorimetric assays

allow for indirect determination of specific concentration such as: proteins, carbohydrates, enzyme activity, etc… via color change.

These reactions can be performed directly inside the

spectrophotometer

.

spectrophotometer

A spectrophotometer: is employed to measure the amount of light that a sample absorbs(absorption) depend on beer lambert law (the intensity of the color and hence the absorption, is directly proportional to the concentration).

Methods for protein determination

Spectrophotometry based on UV absorption:Classical method.Proteins absorb UV light at 280 nm.Dye binding assay (barford assay):Depend on dye which bind to protein and give color.Copper based assays:Lowery (Folin) method.Bicin-choninic acid assay (BCA).Biuret method: for routine use, the biuret procedure is simple to perform, producing a stable color that obeys Beer's Law.All these methods depend on the presence of cupric ions (chelating agent) which bind to peptide bond and give color.Cupric ions (Cu+2) Cuprous ions (Cu+)

reduction

Aim and significance

Aim

:

To perform biuret test for proteins.

To estimate the amount of total proteins in a sample.

To make standard curve.

Significance:

The quantization of protein

content is

important and has many applications in clinical laboratory practices and in research especially in the field of biochemistry. The accurate quantization of protein content is a critical step in protein analysis.

Biuret reagent

Copper sulfate: this provides the cu (II) ions which form the chelate complex.Cu (II) ions: give the reagent its characteristic blue color. Potassium hydroxide (KOH) or sodium hydroxide (NaOH): doesn’t participate in the reaction but provides the alkaline medium.Potassium sodium tartarate (KNAC4H4O6.4H2O):Stabilizes the chelate complex.Prevent precipitation of copper hydroxide.Potassium prevent auto reduction of copper.

Principle

One commonly used method for determining the total protein in a sample is the

Biuret

method.

The

Biuret method is based on the

complexation

of

(

Cu

+2

)

to

peptide bonds

in

the

protein’s.

The formation of a

(

Cu

+2

)

protein

complex requires

two peptide

bonds and produces a

violet-colored

chelate product which is measured by absorption spectroscopy at

540 nm

.

Molecules

containing 2 or more peptide bonds associate with the cupric ions to form a coordination complex that imparts a purple color to the solution with A(max) = 540 nm.

Under alkaline conditions cupric ions chelate with the peptide bonds resulting in reduction of cupric ions to cuprous ions

.

The cuprous ions can also be detected with folin ciocalteu reagent (phosphomolybdic/ phosphotungstic acid), this method is commonly referred to as the lowery method.Cuprous ions reduction of folin ciocalteu reagent produces a blue color that can be read at 650-750nm.The amount of color produced is proportional to the amount of peptide bonds such as size, amount of protein/peptide.

Lowery method

Disadvantages of Biuret test

This

method requires relatively large quantities of protein

for

detection

(

1

- 20

mg/mL

).

It’s

sensitive to a variety of nitrogen-containing substances

as:

bi-urea

,

that

could be in the protein solution, thereby increasing the likelihood of erroneous results

.

Standard curve

A

standard

curve

is a type of graph used as a quantitative research technique.

Standard curve for protein concentration is often created using known concentrations of bovine serum.

The protein we will analyze is

bovine serum albumin

(BSA).

Albumin:

is a serum protein that transports fatty acids and is important in maintaining plasma

pH.

In

protein quantization assays, BSA serves as a

reference protein

that is used to construct protein standard curves

.

Standard solutions

: known concentrations of samples.

Standard curve

The

preparation of a standard

curve is

necessary to check whether the method of assaying a particular substances increases in a linear way with its concentration.

The general formula for obtaining different concentrations of a solution by dilution with

diluent

is:

C1V1 = C2V2

How to calculate the concentration

According to

beers_law

The

Beer-Lambert law (Beer’s law)

mathematically establishes the relationship between concentration and absorbance in many photometric determinations. Beer’s law is expressed as

A =

abc

The concentration of substance is directly proportional to the amount of light absorbed or inversely proportional to logarithm of the transmitted light.

A:

absorptivity

constant for the substance

B: length of the light path through the substance.

Reference range for total proteins is : 66.6 to 81.4

g/l

or 6.6 to 8.1 g/dl.

How to make a standard curve

Multiple samples with known properties are measured and graphed, which then allows the same properties to be determined for unknown samples by interpolation on the graph.

The samples with known properties are the

standards

, and the graph is the standard curve.

Cont….

Draw the points with protein concentrations as x values and the average absorbance as y values on a grid or graph paperDraw a straight line through the pointsLookup the unknown protein concentration from the plot using the absorbance value of the unknown protein.

signal

concentration

Sensitivity: lowest amount of analyte in a sample which can be detected.Specificity: is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Linearity of an analytical procedure: is its ability (within a given range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample.

Procedure

Conc. mg/mlUnknown (ml)Protein standard (ml) D.W (ml)Tubes 000.510.40.10.420.80.20.331.20.30.241.60.40.1520.5060.500unknown

After this, add 1 ml of biuret reagent to each tube and mix. Incubate the tubes for 20 minutes at room temperature.Read at 540 nm on spectrophotometer. Make the standard curve and measure the concentration of unknown.

Blank solution:

is

a solution containing

little to

no analyte of interest, usually used to calibrate instruments such as a colorimeter.