Lab Exercise 0ne

Carbohydrate AnalysisLab A.1Page 26

Biochemical Assay

• Biochemistry deals with the identification and quantification of bio-molecules from a variety of living systems

• Rely on the chemical reactivity and physical properties of bio-molecules to make identification and quantification.

• Primary tool is the spectrophotometer– Uses absorption of mono chromatic light

Spectrophotometer

Measure quantity

• Some bio-molecules have properties which allow direct measurement.– proteins have aromatic amino acids (280nm)– Nucleic acids have unsaturated ring structures

(260nm)• Other molecules have chemical properties

which can be used in indirect measurement.

Introducing concept of standard curve

• Uses dilutions of a solution of known concentration to determine concentration of unknown

A540

[glucose(red)]

b

m = y/x

0

0(may or may not equal 0)

Standard Curve

• Assumes that unknown will respond in assay the same as the known– Valid in todays assay as they (the reactive groups.

glucose) are the same– Problem in other assay as they may not contain

same amount of reactive groups• Protein assays (have to choose)• But usually close

Our model carbohydrate is the sugar glucose

We will exploit its ability to reduce other compounds to produce a product which

can be measured optically

Requirement placed on sugar

• Must be an aldehyde– Ketones and hemiacetal configurations are not

reducing• Conditions of reactions favor conversion to

aldehyde by lowering aldehyde concentration

Sugars as Reducing Agents

Equilibrium between hemiacetal and open chainis driven to open chain as oxidation to acid form takes place. This ensures a quantitative conversion withtime and a stoicheometric production of reduced copper.

Nelson Assay (a two step Rx)

• In the Nelson assay Cu+2 is reduced to Cu+1 by the reducing activity of the sugar (step 1)

• Cu+1 is oxidized to Cu+2 by addition of arsenomolybdic acid (colorless) (step 2)

• Results in blue (reduced) arsenomolybdous acid • Amount is directly related to [CU+1]• Will detect any reducing sugar (concentration of

sugar must be limiting factor)

3,5-dinitrosalicylic acid (DNS) assaySection A1 pages 33-49

• Sugar reduces the organic DNS which absorbs maximally at yellow wave length

• Results in change (shift) in absorption spectrum from yellow to red/brown at 540nm– Different from Nelson reaction

• Measured at 540nm– Unreacted DNS not seen at this wavelength– Amount of absorbance directly related to amount of

reducing sugar

The DNS reagent

From the MSDS:– LABEL PRECAUTIONARY STATEMENTS TOXIC (USA)

HARMFUL (EU) HARMFUL BY INHALATION, IN CONTACT WITH SKIN AND IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. IN CASE OF CONTACT WITH EYES, RINSE IMMEDIATELY WITH PLENTY OF WATER AND SEEK MEDICAL ADVICE.

3,5-dinitrosalicylic acid is reduced to 3-amino,5-nitrosalicylic acid

The DNS assay

• Experimental design and flow charts page 36 &37

• Be sure to read “Hazards” page 37• Protocol on page 38 • Data analysis page 42

Today's Experiment

• Measure the concentration of glucose by detecting the reducing end of the monosaccharide.

• This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced form which absorbs at 540nm.

• Amount of reduced DNS proportional to amount of glucose.

What are we doing today?

Important: See data table page 39

• Pipetting technique is critical to accuracy and to preventing cross contamination of samples– Read Micropipette operation (8 to12)– Pipettes have two stops• First to take up selected volumes• Second to deliver

• Choose pipette “in the range” that you need.

You will create a standard curve

• You are provided a stock solution which contains 1.2 mg/ml

• You will dilute this stock solution in a specified manner always producing a 4 ml solution (See table A1-2)

• After reacting with DNS you will read the absorbance of each solution at 540 and plot vs concentration

• You will compare the A540 of unknown to standard curve

Standard curve

• Uses dilutions of a solution of known concentration to determine concentration of unknown

A540

[glucose(red)]

b

m = y/x

0

0(may or may not equal 0)

Protocol Page 38

• Steps 8,9,10– Critical for uniform reaction rates– 100C accelerates the reaction– Cool samples in Ice water bath for 10 to 15

seconds• Rapidly brings the sample to low temp which slows the

reaction• Carefull too long in ice bath will cause condensation on

the cuvettes

Important

• Careful handling of Cuvettes is essential for accuracy and prevent contamination– Handle only with gloves– Touch only the areas not in the light path– Rinse carefully with DH2O after each use– Always go from lowest concentration to highest

concentration.– Wipe clear surface if necessary with “Kimwipe”

Extremely Important• Put cuvette into Spec slot that is in the beam path • Be certain that clean panes face the beam path• Measure only with the lid closed• Always set the spec with a blank (line 1 table A.1-2, page 39)– Contains all components of reaction except that which is

to be measured– Always use same cuvette

Data collectionTable 1. DNS Assay Calculations

Tube

Number

Water Volume

(ml)

Glucose

“Standard”

Volume (ml)

Glucose

“Unknown”

Volume (ml)

DNS Volume

(ml)

Measured A540 [Glucose]

(mg/ml)

Glucose

Total Amount

(mg)

1(blank) 3.000 0.000 - 1.000

2 2.750 0.250 - 1.000

3 2.500 0.500 - 1.000

4 2.250 0.750 - 1.000

5 2.000 1.000 - 1.000

6 2.750 - 0.250 1.000

7 2.500 - 0.500 1.000

8 2.000 - 1.000 1.000

PLEASE DO NOT SLAM THE SPEC LIDS

Important• 1. Wear Gloves and Safety Glasses• 2. Record the code number of your unknown • 3. Be certain that test tubes are clean• 4. Water/H2O always means distilled water• 5.Have TA initial your data before you leave.

See lab exit requirements page

Application quizAddress in your report

• What does the portable glucometers used by diabetics measure?

• How do they measure it?

Reminder

• Lab Reports are PERSONAL

Grading for This Experiment

• Number of lab periods = 1• Lab Report = 10 points• Pre lab= 3 points• Total = 13 points

Clean up (Please)before you go

• See page 46. Waste Disposal & Clean up

• Return pipettes to rack

Next Lab: Enzyme Kinetics Lab C1Page 73-92. Read carefully

• Due next time: September: 12 & 13.– Prelab assignment for Enzyme Kinetics 1

• Lab report for Carbohydrate Analysis– See Report Requirements page 47-48

Constructing Lab Reports

BCH 452-001

5 Components• (Cover Page)• Abstract• Introduction/Background• Methods• Results• Discussion• (References)

Cover Page• Lab Title• Name• Date• Lab partners• Instructor and TA’s

Abstract• Theory (background/intro and

methods summary)• Results

Introduction• Conceptual Theory• Experimental Theory

Methods• Protocol with general description

• “In a beaker, 5ml of reagent X was mixed with 2ml of reagent Y…” Important statement

• “1) Obtain gloves, lab coat, four micropipettes and a clean beaker . 2) Set a micropipette to 1000μl….”Too much detail, nobody cares

Results• Properly labeled data tables and

graphs• Captions and descriptions• Sample calculations (with units!)• Other requirements? (Percent error)

Graph ExampleThe following graph shows standard curve of glucose concentration. Absorbency readings were taken at 540 nanometers of 5 samples with known glucose concentration. R2 value of .9688 indicates a fit linear correlation. The slope of this graph was used to calculate glucose concentration in unknown samples (Fig 4).

Fig 3: Graph of concentration of “standard” glucose vs. absorbancy at 540 nm for tubes 1-5.

Discussion• Explain why the experiment was run

and what information was gained• Answer questions posed in lab

manual- look at lab report requirements• Results• Sources of error