Nutttttt

LAB 4: PROTEIN METABOLISM

PURPOSE:

To employ proper laboratory techniques in order to determine whether one is in positive,

negative, or zero nitrogen balance by knowing the protein (nitrogen) intake and the

urinary urea nitrogen levels of a 24 hour urine collection.

BACKGROUND:

Proteins are complex organic compounds that are necessary for growth and repair of

all living matter. They are formed from smaller structural units called amino acids. Amino

acids are bonded together to form a "string" which then folds and coils upon itself giving

the protein its characteristic shape and function. There are 20 different amino acids used

in proteins, 9 of which are considered nutritionally essential. Amino acids are all alike in

the respect that each one contains a central carbon atom (C), an amine group (NH2), a

carboxyl group (COOH), and a radical group (R). Proteins differ because the radical group

is different for each amino acid.

H

|

NH2– C – COOH

|

R

When we ingest protein, our body dismantles the dietary protein into its amino acid

components that can be absorbed in the small intestine. Inside the body's cells, the amino

acids are reassembled into proteins specific to our body such as: enzymes, hormones,

structural material, etc.

Although it is considered "biologically expensive", proteins can also be used to provide

energy. When excess proteins or amino acids are ingested, the amino acids that are not

used as building blocks are deaminated. Once the amine group is removed, it is excreted

through the urine as urinary urea nitrogen (UUN). The remaining molecule can enter

catabolic pathways and be broken down into water, carbon dioxide, and energy. If energy

intake from carbohydrates or lipids is sufficient, then excess protein will be stored as fat.

On the other hand, when energy is limiting, the body will use its own protein to provide

energy. Lean muscle tissue and other proteins can be broken down into its amino acids

constituents, deaminated, and ultimately used to provide energy.

Our body is very efficient at conserving and reusing protein, but some is lost through

the feces as unabsorbed dietary protein, through the high turnover rate of intestinal cells,

or through skin, hair and nail losses. Under normal conditions, it is only this small amount

of protein that needs to be replaced daily. The RDA for protein is 0.8 g/kg of body weight

per day.

The need for protein and amino acids is estimated by laboratory analysis but it is not

easy to measure protein in a laboratory. However, it is fairly simple to determine the

nitrogen in protein. Each amino acid contains at least one nitrogen atom and the weight of

the typical protein molecule is 6.25 times greater than nitrogen. By recording the protein

intake and dividing by 6.25, the nitrogen intake can be calculated. The nitrogen output can

also be measured by collecting the urine, fecal matter, and integumentary losses. Highly

controlled metabolic studies have determined that the amount of nitrogen lost through

feces, skin, etc. to be approximately 4g/day. Therefore, it is possible to measure the amount

of nitrogen ingested and the amount excreted. In the healthy adult, the amount ingested

should equal the urinary output plus the approximate 4g/day loss; in other words, a state of

zero nitrogen balance.

The following calculation assists in the determination of nitrogen balance:

Nitrogen Balance = dietary protein – (UUN + 4g)

6.25

In a state of growth (e.g., in infants and children) or repair (e.g., wound healing) the

amount of protein retained in the body exceeds the amount lost. This state is called positive

nitrogen balance. When protein ingestion is lacking, the body dismantles its own protein,

deaminates the amino acids, excretes the nitrogen, and uses the rest of the molecule for

energy. This state is referred to as negative nitrogen balance.

As a result, a nitrogen balance study can be an effective method for estimating protein

needs, but the results can be adversely affected by poor data collection. In order to

maximize the validity of a nitrogen balance study, a high degree of accuracy must be

adhered to when collecting data (e.g., measurement of food intake and collection and

measurement of urine).

I. Preparation of Standard Curve

The BCA™ Protein Assay is a detergent-compatible formulation based on bicinchoninic acid

(BCA) for the colorimetric detection and quantitation of total protein. This method combines the

well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the biuret reaction) with

the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a

unique reagent containing BCA. The purple-colored reaction product of this assay is formed by

the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits

a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a

broad working range (20-2,000 µg/ml). The BCA™ method is not a true end-point method; that

is, the final color continues to develop. However, following incubation, the rate of continued color

development is sufficiently slow to allow large numbers of samples to be assayed together. The

macromolecular structure of protein, the number of peptide bonds and the presence of four

particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible

for color formation with BCA.

Because of the excellent linearity of this procedure, protein concentrations generally are

determined and reported with reference to standards of a common protein such as bovine serum

albumin (BSA). A series of dilutions of known concentration are prepared from the protein and

assayed alongside the unknown(s) before the concentration of each unknown is determined based

on the standard curve. In this exercise you will asked to determine the protein concentration of a

solution.

1. To determine the concentration of your unknown protein sample using the BCA method you

must compare the absorbance of your unknown protein to that of a known protein concentration.

Here we will utilize bovine serum albumin (BSA) to generate a standard curve. See Table 1.

Diluted Standard Volume of protein

standard(2000μg/mL)

Volume of

Diluent (1X

PBS)

Concentration

g/mL

S1 0l 200 l 0

S2 25l 175l 250

S3 50l 150l 500

S4 100 l 100 l 1000

2.votex for 20 seconds to allow the standard samples homogenized.

II. Unknown protein sample preparation 1. To ensure that the absorbance of the unknown protein sample falls within the standard

curve, you may need to prepare dilutions of your unknown.

i. You will prepare seven dilutions, see Table 2. You will determine what dilutions to make for three samples. Use Table 2 to complete your calculations.

ii. Before making the dilutions, please have an instructor check your calculations

Table 2 Dilution of unknown sample

Name Volume of

unknown sample

(µl)

Volume of

diluent (1X

PBS) (µl)

Dilution

factor

Total

volume

U1 200 0 1 200µl

U2 100 100 2 200µl

U3 50 150 4 200µl

III. Protein assay

Option 1. Semi-micro cuvette with spectrophotometer

1. ) Add 1.8 mL of working reagent (WR) to each of 200-µl dilutions (S1-S4 and U1-U3), bring the tubes up to 2.0mL.

2. ) Cover all tubes, than vortex and mix well for 10 seconds, and incubate in 37°C for approximately 30 minutes. Once time is up, be ready to read the concentration. Cool the

plate to room temperature (RT; 5 minutes on the bench top).

3. ) Turn on the spectrophotometer and set measuring wavelength to 562nm: Make sure you pick the correct cuvette (1 mL)!

4. ) Transfer 1 mL solution into the cuvette (duplicate ) 5. ) Measure and record the absorbance of the all tubes in reverse order and rinse clean the

cuvette each time after measurement (Try completing the measurements within 10-15min).

Results:

1. Absorbance reading

Tub

e

absorbance reading at 562

nm

average absorbance

reading

B 0.00 0.00

S1

S1

S1

S2

S2

S2

S3

S3

S3

S4

S4

S4

S5

S5

S5

U1

U1

U1

U2

U2

U2

U3

U3

U3

U4

U4

U4

2. Construction of a Standard Curve: 1) Plot Absorbance vs concentration (µg/mL) for the BSA standards S1 through S4 to obtain

the standard curve using MS Excel. (Do not forget to standardize absorbance with S1

before making a linear regression line in excel!)

2) Use the curve fitting options to determine the linear least squares fit of the standard curve data, display the equation on your graph.

3. Unknown Protein Concentration Determination 1) Determine the trend line equation and use that to calculate the concentration for your

unknown sample.

2) ONCE YOU OBTAIN A STANDARD CURVE: OBTAIN y= mx + b formula than solve for (X). Your unknown reading goes in the Y and student should solve for X.

3) If the absorbance value of unknown samples are out of the standard value range, then they cannot be used to solve for X. Remember to multiple its diluent factor if you use a

diluted unknown sample to solve for X.

4) If there are more than one unknown sample fall in the standard value range, only one unknown sample value is needed for calculation.

4. Lab Report 1.) Follow the report format described in assignment 3.

2.) Results should include absorbance reading, standard curve, formula, and unknown

sample determination

3.) Description of graph in paragraphs

4.) Report the original unknown sample’s value.

5.) Concepts to consider for discussion:

Discuss problems that were associated with the lab (if any).

Show calculations: How do you calculate concentration of unknown from graph

and from formula given?

Interpret the meaning of value for unknown sample