Mohd Hairi Bin A.Hamid Bsc.

Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Introduction Protein Protein found in all cells, body fluids and plasma. High blood, serum, plasma, lymph nodes and some exudates. Low spinal fluid Trace urine. Proteins are compounds, containing carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sometimes sulphur (S). Protein molecules are made from a lot of amino acids molecules, and joined together by peptide bonds.

Peptide bond

Levels of protein as follows:

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Functions of protein are: Antibodies (Immune system) Transport: Lipids, vitamins and toxic substances Coagulation factors (Fibrin; Fibrinogen) Regulation of metabolism Catalyse the metabolic reaction (Enzymes) Hormones (Insulin, Glucagon) Chromosomes (DNA, RNA) Nutrient Protein source (Fishes)

There several reasons why each specimens are measured to determine the total protein: Reasons to measure Provide general info (nutritional status, presence of severe disease in liver, kidney, bone marrow Evaluate glomerular integrity, renal tubular function, presence of overflow of abnormal plasma protein Evaluate blood brain barrier integrity, CNS protein synthesis, CNS infections Provide data to differentiate transudate from exudate

Types of specimen Serum Urine CSF Body Fluid

Protein are usually colourless, therefore it is difficult to measure the presence of protein So, easy method is by staining the protein with Biuret reagent and measure the amount of absorption of the Biuret reagent by a spectrophotometer

Biuret test Biuret test is used to detect the water soluble protein in the samples. It is detected by presenting of peptide bonds Biuret reagent consists of sodium hydroxide (NaOH) and Copper Sulphate (CuSO4). The blue reagent will convert to violet when protein present.

Blue

Purple

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

When peptide bond exists, copper (II) ion will reduce to copper (I) ion, which is violet colour in alkaline CuSO4 solution.
Copper (II) ion (CU2+) (Blue) Copper (I) Ion (Cu+) (Violet)

A violet colour indicates the presence of proteins Biuret test can be Qualitative or Quantitative Test. Qualitative measure the intensity of colour produced by spectrophotometer Quantitative more protein present more darker the colour

Beer-Lambert Law This test principles is according to Beers Law and Beer-Lambert Law Beers Law state that the amount of light absorbed is directly proportional to the concentration (protein) More concentrate, more absorbed, high absorbance Less concentrate, less absorbed, low absorbance While, Beer-Lambert Law said that the intensity of the colour is directly proportional to the concentration (protein) More condense the colour, high concentration of protein present More light the colour, low concentration of protein present

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Biuret Protein Assay

Principles Serum proteins will form a violet complex with copper ions (Cu+) in alkaline CuSO4 solution. The absorbance of the complex is measured at 540 nm.

Equipment and materials 1. Standard: Protein (5 g/dL) 2. Distilled water (dH2O) 3. Biuret Reagent (Copper Sulphate, Potassium sodium tartarate, Potassium iodide, Sodium Hydroxide) 4. Various Protein sample (Concentration: 10, 20, 30, 40 g/dL) 5. Micropipettes (20 l and 1000 l) 6. Cuvette: 1 cm optical path 7. Room temperature (20 25C) 8. Spectrophotometer (wavelength: 540 nm) 9. 6 Test tubes 10. Tube rack

Specimen type, collection and storage Specimen : Serum Storage: Stable at 0 - 4C Avoid hemolysis, fasting specimen is preferred to avoid lipemia Separate the blood as soon as possible (ASAP); early.

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Procedure Example: 1. By using micropipette (50l and 1000l), pipette into the test tubes as follow: Protein Volume and concentration 20 l (5 g/dL) 20 l (10 g/dL) 20 l (20 g/dL) 20 l (30 g/dL) 20 l (40 g/dL) Distilled Water 20 l Biuret Reagent 1000 l 1000 l 1000 l 1000 l 1000 l 1000 l

Blank Standard Tube 1 Tube 2 Tube 3 Tube 4

2. All the substances were mixed. The blank, standard and tube 1 - 4 were incubated for 10 minutes at 20 25C (Room temperature) 3. After 10 minutes, by using spectrophotometer (540 nm); the absorbance of the blank was read first. After autozero, absorbance of standard, tubes 1 - 4 against blank was read and recorded in the table. Results Example: Standard Tube 1 Tube 2 Tube 3 Tube 4 Calibration Although measurement of total protein is simple, calibration is VITAL to implement to ensure the method is under Technique. Some calibration includes: Use Bovine Serum Albumin (BSATM) as calibrator Use Lyophilized Serum Calibrator (Eg: Human serum albumin) Prepare CALIBRATION CURVE (check linearity of spectrophotometer) If linear, can use single standard routinely Repeat the calibration curve at least once a month
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Absorbance 0.08 0.10 0.20 0.40 0.80

Protein Assay

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Preparation of standard curve From the result obtained, plot the data with Protein concentration on the horizontal axis (X-axis) and Absorbance on the vertical axis (Y-axis).

Y-Axis

Intersection

Abs = 0.55

Linear progression

Protein concentration 30 g/dL X-Axis

From the graph also we can predict the concentration of protein with any known Absorbance For example: Absorbance is 0.55.....so, what the protein concentration?? So, the method to determine protein concentration showed on the graph above ( )

Calculation Total protein [g/dL] = Abs. Sample Abs. Standard X Conc. Standard [g/dL]

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Reference ranges Ages Adults Children 1 30 day (s) 1 6 month (s) 6 months 1 year 1 18 year (s) Malaysia reference range Normal total protein level : 6.2 8.2 [g/dL] Female 4.2 6.2 4.4 6.6 5.6 7.9 5.7 8.0 [g/dL] 6.6 8.8 Male 4.1 6.3 4.7 6.7 5.5 7.0 5.7 8.0

Other method to determine protein Bradford Protein Assay Is a colorimetric protein assay to determine total protein level in a solution, and based on absorbance shift in the Coomassie Blue G-25 dye. Protein Red (470nm) H+ Green (650nm) H+ Blue (590nm) Blue-Protein (590nm)

At the begin dye is (Red Form) and after bind with protein (Blue form).

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Lowry Protein Assay Is a colorimetric protein assay to determine the total level of protein in solution Colour change of the sample solution is directly proportional to protein concentration Best used with protein concentrations of 0.01-1.0 mg/mL Based on the reaction of Cupric Ions (Cu+) with peptide bonds, with Folin reagent (Phosphotungstic acid) Under alkaline condition (Like Biuret Test) Cu2+ + Peptide Bond = Cu+ Cu+ + Radical Group (Tyrosine, tryptophan, cysteine) + Folin reagent = BLUE

Summary Biuret Protein Assay (BCATM Protein Assay) Features & Advantages Working reagent stable for 1 month at RT Compatible with reagent Simple, easy to perform Less Protein variation than Coomassie dye method Work with peptides (3/more amino acids) Flexible incubation protocol Good Accuracy and convenience Measure amount of protein Not compatible with thiols/ reducing agent Not a true end-point assay Reducing agent (Ammonium salt) Thiols, ascorbic acid, uric acid

Application Disadvantages

Interferences

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Bradford Protein Assay Features & Advantages Application Simple to perform One-reagent system, stable for 12 months No filtration or dilution needed Fast ; total preparation & assay time < 30 minutes Colour response sensitive to pH Temperature-dependent colour response Compatible with buffer salts, metal ions, reducing agents, chelating agents Have protein variation with Coomassie blue dye Good accuracy & Excellent convenience Standard assay Micro assay Protein recovery studies Microplate format assay Nonlinear colour response Protein must be > 3000 Da Microassay has potential for interference Commonly none Sometime detergent. Lowry Protein Assay Features & Advantages 2 reagent system 2 step incubation required Colour response read at 750 nm Work with peptides (3/more amino acids) Less protein variation (like Biuret assay) Good accuracy & Fair convenience Most cited protein assay in literature Adaptable for use with microplate Longer total assay time Timed addition of Folin reagent adds complexity Practical limit of about 20 samples per run Detergent Thiols, disulphides Copper chelating reagent Glycerol, Tricine Strong acids, ammonium sulfate

Disadvantages

Interferences

Application

Disadvantages

Interferences

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Clinical significance Causes of alterations of serum total protein includes: Change in volume of plasma water Change in concentration of specific protein in plasma Hyperproteinemia increase protein concentration, decrease plasma water volume in plasma Hypoproteinemia decrease protein concentration, increase plasma water volume in plasma The measurement of total protein level can be used to diagnose and detect: Intestinal malabsorption Nutritional deficiency Impairment of kidney function Defectiveness of protein synthesis in the liver Chronic inflammatory disorder Liver cirrhosis Dehydration Increased total protein Dehydration Monoclonal disease Multiple myeloma Macroglobulinemia Cryoglobulinemia Chronic polyclonal disease Liver cirrhosis Sarcoidosis SLE Chronic infections Decreased total protein Protein loss Nephrotic syndrome Severe burn Protein-losing enteropathies Failure of protein synthesis Starvation Protein malnutrition Severe non-viral liver cell damage

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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

References 1. Meliyanti M.Gunatillaka et al (2005), Manual on Standard Operation Procedures, Sample Collection, and Reference Range for Clinical Chemistry: Total Protein, WHO Ministry of Health and The Department of Biochemistry, Medical Research Institute, Sri Lanka. 2. CSDS for Total Protein FS* : diagnostic reagent for quantitative in vitro determination of albumin in serum or plasma on photometric systems (December 2008), Diagnostic system; received on January 01, 2010 3. Mary Hotaling (1998), Saunders Manual of Clinical Laboratory Science (1st Edition): Amino Acids and Proteins, W.B Saunders Company. 4. Bishop, M. L., etal. (2000). Clinical Chemistry: Principles, Procedures, Correlations (4th ed.). Philadelphia, PA: Lippincott Williams & Wilkin 5. Protein Assay Technical Handbook (2005), Pierce Biotechnology, Inc retrieved on February 17, 2010 from http://www.piercenet.com/path95n 6. Biuret Test (January 31, 2010), retrieved on February 10, 2010 from http://en.wikipedia.org/wiki/Biuret_Test 7. Protein Assay (1998), retrieved on February http://bioweb.wku.edu/courses/biol121/Protein/Biuret 11, 2010 from

8. David R. Caprette (October 04,2005), Hartree-Lowry and Modified Lowry Protein Assays, retrieved on February 11, 2010 from http://www.ruf.rice.edu/%7Ebioslabs/methods/protein/lowry.html

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