Lab Exercise One

Carbohydrate Analysis Lab A.1

2003

Biochemical Assay

   Biochemistry deals with the identification and quantitation of bio molecules from a variety of living systems Rely on the chemical reactivity and physical properties of bio-molecules to make Identification and quantitation.

Primary tool is the spectrophotometer

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.

We will do the DNS assay Section A1 page 4    Measures the reducing capability of glucose Uses a color conversion reaction from yellow to red brown A 540 Conversion of moles DNS equals moles of glucose.

Introduce concept of standard curve  Uses dilutions of a solution of known concentration to determine concentration of unknown

A 540 b

(may or may not equal 0) 0 0

[glucose (red) ] m =



y/



x

Standard Curve

 Assumes that unknown will respond in assay the same as the known   Valid in DNS assay as they 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

Reducing Sugars

   Have aldehyde group Can be oxidized to acid Reduces another compound

Requirement placed on sugar

  Must be an aldehyde  Ketones and hemiacetal configurations are not reducing Conditions of reactions favor conversion to aldehyde

Sugars as Reducing Agents

Equilibrium between hemiacetal and open chain is driven to open chain as oxidation to acid form takes place. This ensures a quantitative conversion with time 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

The DNS assay

   Experimental design page 7 Protocol on page 8 section A4 Data analysis page 10

3,5-dinitrosalicylic acid (DNS)    Sugar reduces the organic DNS which absorbs maximally at yellow wave length Results in change (shift) in absorption spectrum from red/orange to red/brown 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

Other Assays (page 4 to 7)     Mucic Acid test   Detects the presence of Galactose Produces insoluble saccharic acid Elson-Morgan test   Detects amino-sugars (Hexosamines) Produces a color reaction Seliwanoff test   Specific for ketose Color reaction Bial’s test  Specific for pentose  Color reaction

Lab reports for this class (see Report construction Page 12)

    Abstract. Statements regarding:  WHAT you are doing (-> procedure)  WHY you are doing it (-> your hypothesis)   WHAT you hope to accomplish (-> also hypothesis) Cf. ‘purpose/goal’ in a good lab notebook! Might think of it as a very short introduction.

Results/Data/Data Analysis Discussion MUST relate data analysis to hypothesis!

We chose this format to try to make the workload manageable and quality-oriented.

Next week: amino acid titration page 53 Lab B.3

 Due next week:   Prelab assignment for Titration of an Amino Acid Lab report for DNS assay    Abstract Data table, graph with best-fit line, calculate ave conc of unknown and std dev in average.

Discussion: linear relationship? Can also use ‘example questions’ as topics for discussion.

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.

Important: See data table page 8   Pipetting technique critical to accuracy and to preventing cross contamination of samples  Pipetters have two stops   First to take up selected volumes Second to deliver Choose pipetter “in the range” that you need.

Important

 Careful handling of Cuvettes essential for accuracy and prevent contamination  Handle only with gloves     Touch only the frosted area Rinse carefully with DDH 2 O 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 clear panes face the beam path Measure only with the lid closed Always set the spec with a blank  Contains all components of reaction except that which is to be measured

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/H 2 O always means distilled water 5.

Have John or I initial your data before you leave

Spectrophotometer

Shimadzu UV-VIS Spectrophotometer

  Wavelengths 200-800 nm Select wave length needed.

Grading for This Experiment

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

Next Week: Ionic Properties of Amino Acids

 Read ExperimentB.3 page 53