Serum Protein

Course Outline
Lecture Notes
Readings
Assay Manual
Experiments
Assays

Laboratory 3: Biuret Serum Protein

Background:

There are three recognized and widely used methods to determine protein concentrations. Each differ in their sensitivity and specificity to measure proteins in solution. We use the Biuret method, which is the predominant method used to measure serum protein. The Biuret method is a colorimetric procedure, which can be purchased from Sigma, or be easily made from a mixture of six chemicals. So that you can work on your lab skills, you are to make your own solution.

This lab will also require you to apply knowledge of dilutions in the preparation of a standard curve, and to become more familiar with the use of the spectrophotometer.

Part I. Preparation of the Biuret Reagent.

The Biuret reagent is based on a reaction between copper and molecules with at least 2 peptide bonds. The color intensity caused by this reaction is directly proportional to the number of peptide bonds, and therefore the amount of protein.

The reagent can be prepared as follows;

Compound	Amount (g)	Instructions
Potassium sodium tartrate	4.5	Dissolve in 40 mL of 0.2 N NaOH
Copper sulfate	1.5	Add to above
Potassium iodide	0.5	Add to above
		Dilute the above to 500 mL using 0.2 N potassium iodide in 0.2 N NaOH

Note: You have to prepare your own regent for this lab.

Part II. Standard Curve - Calculations of unknown protein solutions.

Experimental Procedure:

Using the protein standard solution you will prepare a five point standard curve by performing serial dilutions between a range of 0-150 mg/mL. Use these wit the Biuret reagent to quantify serum protein for the blood samples from incremental exercise to fatigue.

Materials

	Biuret reagent
	calibrated pipette (0-50 mL) and tips
	12 x 75 mm tubes
	tube stoppers
	standard (Bovine Serum Albumin - BSA) and unknown (rest and exercise) protein solutions
	spectrophotometer

Methods

1. Add 3 ml of Biuret reagent to an appropriate number of 12 x 75 mm glass tubes.

2. Pipette 50 mL of each sample from your serial dilutions, from the incremental exercise samples, and for distilled water (blanks) into the tubes – make sure you perform duplicate samples. Vortex all tubes. Incubate the tubes at room temperature for 30 min.

3. During incubation, turn on and warm-up the spectrophotometer.

4. Prepare to run all samples at 540 nm, and record the absorbance values for the standards and unknowns. You can either record the absorbance values for the blanks, or zero to them.

5. Calculate the serum protein concentrations using the regression equation from your standard curve.

Data Analyses:

	When using a standard curve, why are no dilution corrections required?
	What are typical values for serum protein?
	Calculate the change in plasma volume based on a percent change in serum protein. Graph this data, and compare to the hemoglobin and Hct determined %PV changes (Dill and Costill method).
	What physiological conditions may alter serum protein concentrations?
	Briefly (1 page) discuss some research that has used serum protein concentrations to evaluate fluid shifts in the body.

References

Costill D.L. and K.E. Sparks. Rapid fluid replacement following thermal dehydration. J. Appl. Physiol. 34(3):299-303, 1973

Sjogaard G., R.P. Adams and B. Saltin. Water and ion shifts in skeletal muscle of humans with intense dynamic knee extension. Am. J. Physiol. 17:R190-R196, 1985.

Convertino V.A., P.J. Brock, L.C. Keil, E.M. Bernauer and J.E. Greenleaf. Exercise training-induced hypervolemia: role of plasma albumin, renin, and vasopressin. J. Appl. Physiol. 48(4):665-669, 1980.

see also JAP website for recent abstract to be published in March issue:

Lundvall J and P. Lindgren. F-cell shift and protein loss strongly affect validity of PV reductions indicated by Hb/Hct and plasma proteins. J. Appl. Physiol. 84(3):822-829, 1998.