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BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

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BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

PROTEINS QUALITATIVE ANALYSIS OF PROTEINS


- Biomolecules that contain many amide bonds (peptide bonds) Experiment 1: Isolation of Casein from Milk (Acid/Base
formed by Amino acids Hydrolysis & Neutralization)
- 40 naturally occurring proteins exist Milk
- 20 40 naturally occurring amino acids exist - Good source of calcium & phosphorus but lacks iron and
- Levorotatory form is the natural occurring protein vitamin C
- Left - N-terminus (amino group); Right - C-terminus - Contains 3.4% proteins (Casein = milk protein)
(carboxyl group) Casein
- 50% of the dry weight of the human body - Major protein in milk {complete protein}
- pH of milk = 6.6 - Globular storage protein
- 2 types based on shape: (1) Globular & (2) Fibrous - All Special AA (M, I, L, K, F, R, H, W, T, V)
Dipeptide = 2 AA + 1 P-bond - heating at 55C = facilitates precipitation
Tripeptide = 3 AA + 2 P-bonds - has phosphate groups attached to the hydroxyl groups of
Polypeptide = more than 3 AA some AA side chains = Phosphoprotein
Protein Functions: - Isolation using ISOELECTRIC PRECIPITATION =
1. Structural - collagen & keratin lowering pH up to isoelectric point (Isoelectric pH with
2. Contractile - myosin & actin zero net charge)
3. Transport - hemoglobin (4) & lipoprotein - IpH = zero net charge - Protein is most soluble
4. Storage - casein & ferritin - Calcium caseinate (calcium salt in casein) has an IpH of
5. Hormone - insulin & growth hormone 4.6
6. Enzyme - sucrase & trypsin - 10% Acetic Acid - lowers the pH & used as precipitating
7. Protection - immunoglobulin agent
Structural Level: Hydrolysis
1 (Primary) - Amino acid identity = peptide bond (C.B.) - Breakage of the peptide bond in the presence of H2O &
2 (Secondary) - -helix & -pleated sheet = H-bonds Catalyst (Acid, Base or Enzyme)
(N.C.B.) - 2 Types: (1) Complete - Acid & Base hydrolysis; (2)
3 (Tertiary) - IMF between side chain , 3D Structure Incomplete - Enzyme hydrolysis
(C.B.) = disulfide bonds of Cysteine - Acid Hydrolysis = 8N H2SO4 (16 molar) -> dilute
(N.C.B.) = H-bonding, Hydrophobic Interaction of solution = presence of H2O {conc. H2SO4 = 18 molar}
Valine, - complex, Ionic bond, Metal-ion - Base/Alkaline Hydrolysis = Ba(OH)2 (s)
coordination bond
4 (Quaternary) - for oligomeric protein (multipeptide
chains)
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

2.0 4.6 6.6 7.0


7.0

Insoluble IpH of Casein - most insoluble pH (Isoelectric Pt.) pH of Milk Soluble

Acid Hydrolysis Base Hydrolysis


o Similarities: (1) autoclaved @ 15 psi for 5 hours; (2) filter (filtrate = Hydrolyzate contains only AA)
o 8N H2SO4 instead of HCL: Ba(OH)2 (s) instead of NaOH:
HCl + Ba(OH)2 BaCl2(aq) + H2O NaOH + H2SO4 Na2SO4(aq) + H2O
Neutralized with Ba(OH)2 BaSO4 Neutralized with H2SO4 BaSO4
Advantages: Advantages:
No Racemization - all AA retain their structural - configuration W is not destroyed
Disadvantages: Disadvantages:
W is destroyed, forming Humin (black color) Racemization/ formation of racemic mixture (both L & D)
loss: S& T loss: S, T, C
N&Q D&E R urea & ornithine

Neutralization
- To remove the excess acid/ base in the reaction
- A chemical reaction in which an acid & a base interact with the formation of salt and a H2O by-product
- Acid H. Neutralization = Ba(OH)2(s), Ba(OH)2(aq) , 7mL H2O
- Base H. Neutralization = 16N H2SO4, 8N H2SO4, 7mL H2O

Percent Yield = (weight of casein weight of milk) x 100


1mL = 20 drops

Experiment 2: Color Reaction of Intact Casein & Hydrolyzate


Intact Protein
- Individual AA are linked together by peptide bond (very strong)
Hydrolyzate
- Product of hydrolysis; peptide bonds undergone cleavage
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Color Rxn Test Test for Reagent(s) Principle Positive Results Other Information
Negative for Both
Violet solution = color
General test for Intact CuSO4 Hydrolyzate
Complexation reaction intensity based on
Biuret Test Proteins (presence of 5 H2O (N.R. = urea,
(Cu+2 & amide N atoms) peptide bond length
peptide bonds) NaOH dipeptides &
(Intact)
coagulated proteins)
Negative for:
Violet or Deep Blue P & OH-P (cyclic
General test for free AA Ninhydrin = Oxidative Deamination &
Solution imino acid)
Ninhydrin Test (presence of - amino triketohydrindene Condensation (Reduction
(All Hydrolyzate & = yellow
groups) hydrate in ethanol of Ninhydrin)
Ammonium salts) Amine (aniline)
= orange to red
conc. HNO3 Nitration of Aromatic Presence of electron
General test for Aromatic Yellow ppt to Orange
Xanthoproetic Test conc. NaOH (neutralize rings via Electrophilic donating substituents
AA (W, F, Y) soln (Alkaline)
excess acid) Aromatic Substitution enhances reaction rate
Glyoxilic Acid (Mg +
Acid catalyzed- Reduction of Oxalic
Specific test for Indole Oxalic Acid)
Hopkins-Cole Test Condensation & Violet Interphase Acid to Glyoxilic
Group ( W ) conc. H2SO4
Complexation Acid
(dehydrating agent)
Urea is used to
-napthol (condensation) Base catalyzed-
Specific test for Guanido Red coloration which stabilize color &
Sakaguchi Test NaOH (catalyst) Condensation &
Group ( R ) easily disappear destroy excess OBr-
NaOBr (oxidizing agent) Complexation
anions
Specific test for Phenolic *Hg(NO3)2
Millons Test Complexation Flesh/ Red ppt -
Group ( Y ) conc. HNO3
Specific test for *Pb(Ac)2 Degradation &
Lead- Acetate Test Black ppt (lead sulfite) -
Sulfuhydril Group (M,C ) NaOH Substitution
*Heavy Metal = non-environmental element (color reaction is not done)
Color Reaction Test Intact Proteins Acid Hydrolyzate Base/Alkaline Hydrolyzate
Biuret Test YES NO NO
Ninhydrin Test NO YES YES
Xanthoproetic Test YES YES YES
Hopkins-Cole Test YES NO YES
Sakaguchi Test YES YES NO
Millons Test YES YES YES
Lead- Acetate Test YES YES YES
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

QUANTITATIVE ANALYSIS OF PROTEINS


Experiment 3: Bradford Assay Method - Pinkish brown (protein unbound) = 465 nm
Bradford Assay Method - Blue (protein bound) = 595 nm - anionic form
- 20-220g protein (required weight) = Bovine Serum Reagent Blank
Albumin (from cow) - contains everything except protein sample
- Uses the Coomassie Brilliant Blue G-250 dye - To tare / auto-zero
- Intensity of color (absorbance) is directly proportional to Unknown concentration is measured using Linear
protein concentration = Beers Law (A = bc) Regression Analysis: y= mx + b
Coomassie Blue Brilliant G-250 o where: y = measured absorbance; m = slope/ B ; x =
- binds electrostatically in anionic form with basic AA side unknown conc; b = y-intercept/A
chains (R, H, K = ionic interaction - sulfate group) and - For standard protein preparations, use C1V1=C2V2 when
stacking with aromatic AA (hydrophobic interaction - non dilutions are done on standard solutions
polar)

SAMPLE PROBLEM:
1 2 3 4 5 6
mL stock solution 0 2 4 6 8 9
mL H2O 10 8 6 4 2 1
mL dye 5 5 5 5 5 5
concentration (g/mL) = x 0 40 80 120 160 180
A595 = y 0.0 0.1296 0.1259 0.3321 0.4611 0.4913

C2 = C1V1 V2
Where: C2 = 40 (x)
Equation of the line: y= mx + b
C1 = 200 (standard stock solution)
A = b = 6.07 x 10-3
V1 = 2 (mL stock solution)
B = m = 2.76 x 10-3
V2 = 10 (constant = sum of stock soln & H2O)
y = 0.1563 (given)
HOW TO: 0.1563 = 2.76 x 10-3 (x) + 6.07 x 10-3
0.1563 - 6.07 x 10-3 = 2.76 x 10-3 (x)
1. Mode - Stat - 2(A+Bx) - Input Data for X (conc) & Y
x = (0.1563 - 6.07 x 10-3) / 2.76 x 10-3
(abs)
x = 54.51 g/mL (1000) = 0.05451 mg/mL
2. Shift - Stat - 5(Reg) - for A/b & B/m value
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Experiment 4: Salivary Amylase Enzyme Inhibitor


Enzymes - Inhibitor bonds to the E to alter or destroy the Es activity
- Biological catalysts for chemical reactions in all living organism - can be reversible or irreversible
- increases that rate of reaction (106 to 1012 times faster) = reusable - Inhibitor Types:
& remain unchanged; lowering of activation energy Noncompetitive Inhibitor
- are 3D polypeptide capable of adapting a unique shape (active o bonds to the E but not to the AS
site) into which a substrate possessing similar shape can fit o When this inhibitor is bound, the AS changes shape and
- very specific; in terms of the type of reaction & affected by the the S no longer fits
following (1) pH, (2) temperature and (3) substrate concentration Competitive Inhibitor
- operates at a very high speed o has a shape & structure similar to S, so it competes with
- 2 step reaction process the substrate for binding to the AS
- includes a Cofactor = a metal ion/ organic molecule needed for
enzyme - catalyzed reaction to occur Enzyme Classification
Class Class Name Catalyzed Reaction
Enzyme Mechanism #
I. An enzyme contains an active site that binds the substrate = transfer of e- = Hydride ions (redox
1 Oxidoreductases
forming an enzyme - substrate complex rxn)
II. Once the reaction has occurred, the catalyst released the 2 Transferases group transfer rxn
products transfer of FG to H2O (hydrolysis
3 Hydrolases
III. Specificity occurs as explained by the following models: rxn)
A. Lock and Key Model (1) addition of groups to double
bonds
o both components (enzyme & subs) fit each other to a 4 Lyases
(2) formation of double bonds by
very high degree
removal of groups
o shape (exactly the same with AS) of the S fits into the
transfer of groups within molecules
binding/active site of E 5 Isomerases
to yield isomeric forms
o Complementary Shape occur naturally formation of C-C, C-S, C-O & C-N
o Active Site = rigid bonds by condensation rxn coupled
B. Induced Fit Model (Glove and Hand Model) 6 Ligases
to cleavage of ATP or similar
o the flexibility of the enzyme to conform cofactor
o binding of S to the E is due to the ability of the E to
adjust and be flexible
o Complementary Shape forms after binding
o Active Site = flexible
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Naming Enzymes Starch Hydrolysis


1. Trivial o conversion of starch to dextrin, maltotriose, maltose & glucose
2. Subs + -ase o Enzymatic hydrolysis of starch breaks large, insoluble starch
3. Action + -ase molecules into soluble (in H2O) starches:
4. Combo + -ase Amylodextrin
5. Numerical - gives blue color in I2 solution
Erythrodextrin
Amylase - gives red color in I2 solution
o enzyme that breaks down starch into sugars Achrodexrin
o class # 3 - Hydrolases enzyme - gives no color in I2 solution = salivary amylase is active
o found in saliva = begins the chemical rxn of digestion
Starch
- Amylase o white, tasteless, solid carbohydrate
o found in starch o has linear amylose & branched amylopectin
o also called - (1-> 4) - D - glucan - 4 - gulcanohydrolase" o chemical formula: (C6H10O5)n
or glycogenase o converted to glucose as a source of energy for organisms
o are calcium metalloenzymes completely unable to function o can be detected by the blue-black color when I2 solution is
in the absence of calcium added
o both salivary & pancreatic amylases belong to this type o Yellow Solution = complete hydrolysis/ disappearance of
o breaks down carbohydrates yielding: starch
o Amylose
- has linear - (1-> 4) G.L. Basis for the Rate of Reaction of Salivary -Amylase
- produces glucose and maltose 1. Disappearance of Reactant (Starch)
o Amylopectin - disappearance of blue-black color given by the starch-
- has linear - (1-> 4) & branched - (1-> 6) G.L. iodine complex
- produces glucose, maltose and dextrin - complex formed by complexation of amylose with an
iodine polymer where triiodide (I3-) forms by reacting
Salivary Amylase iodine (I2) with iodide (I-)
o also called EC 3.2.1.1 2. Determination of Achromic Point
o digestive enzymes = -amylase - time taken by S.A. to hydrolyze starch to achrodextrin
o -amylase - utilize starch as a substrate and produces at optimum conditions
reducing sugars as products: Maltose (dissac.) & Dextrin
(oligosacc.)
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Factors Affecting Enzyme Activity A. pH


I. Enzyme & Substrate Concentration Constant: 37C = normal body temperature
- Inc. rate of rxn = Inc. S conc & constant E conc Variable: pH Buffer - 4, 5, 6.7, 8, 10
- Inc. the # of S molecules = Inc. probability that S o Optimum pH: 6.7 (pH of saliva of a healthy individual = 6.5)
will collide with E to form an E-S complex. (E is o will affect the polar & non-polar intramolecular attraction &
excess) repulsive forces and interaction between E & S
- At a certain conc, the rate of rxn reaches the o affect the conformation of the enzyme
maximum level o Acidic/ Basic Medium = denatured enzyme (change in
conformation -> 3 structural level)
II. Inhibitors & Activators o From lower pH E activity will increase until Optimum pH then
- non-competitive & competitive inhibitor after E activity decreases
o At Optimum pH, the S attaches itself to the E via 2 ionic bonds
III. pH & Temperature o Ionic bonds are caused by the transfer of a H ion from a -COOH
Constant Factors: group in the side chain of AA1 residue to an -NH2 group in the
Starch - substrate side chain of AA2
Amylase - enzyme o At Lower pH, the -NH3+ group will not be affected, but the -
NaCl - to mimic normal physiological condition in COO- will pick up a H ion (protonation) = inability to form
the body (amylase needs Cl- to be able to perform its ionic bonds -> enzyme is inactive
function) B. Temperature
Constant: 6.7 = Buffer pH
o Chloride Ion (Cl-) Variable: Temperature (C) - 4, RT, 37, 50, 60, 70
- An allosteric effector (selectively binds to a protein & Rate of Rxn = 1/ t or min-
regulates its biological activity) o Optimum Temp: 37C (max amount of products formed)
= Allosteric Regulation - regulation of enzyme by o At Low Temp, amylase is ineffective = Inactive
binding an effector molecule at the proteins allosteric site o At High Temp, amylase is less effective = Thermal Denaturation
o reaction rate increases proportionally with the rise of temp
- acts a ligand that can increase or decrease enzyme activity o proportionality due to increase in kinematic energy which
increases frequency of collision of E & S
- -Amylase binding site has positively charged amino o inc in temp = inc amount of energy available for the reactants to
acid residues that assists in the binding of negatively reach its transition state = E-S complex
charged Cl- = causes conformational change to the enzyme o the rate of most E rxns approx. double for every 10C temp rise
switching it to the more active state o When E denatures, conformation or struc. alters irreversibly
o inc in thermal agitation break down the 2 & 3 level that rely
on H-bonds, VdW forces & ionic bonds = alters shape of AS
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Experiment 5: General & Specific Test for Carbohydrates Classification


Carbohydrates 1. Number of C atoms ( e.g. triose = 3C; pentose = 5C)
- called sugars and starches
- most abundant class of organic compounds found I 2. Nature of reactive carbonyl group
- polyhydroxy aldehydes or ketones = compounds that can be Aldoses - aldehyde group containing
hydrolyzed to them Ketoses - ketone group containing
- general formula: CnH2nOn (n 3)
- are synthesized in green plants by photosynthesis = energy 3. Number of Saccharides / Hydrolysis Activity
from the sun is stored as chemical energy A. Monosaccharides
- serve as structural material o the simplest carbohydrates
- component of energy transport compound ATP (ribose) o Generally have 3 to 6 C atoms in a chain with an aldehyde or
- one of 3 essential components of DNA & RNA ketone ending and many OH groups.
- used for bursts of energy needed during exercise in the form o Simplest Aldose (aldotriose) is Glyceraldehyde and the simplest
of glucose in the body Ketose (ketotriose) is Dihydroxyacetone = constitutional
isomers (C3H6O3)
o cannot be broken down by hydrolysis

Name Structure Description

Xylose Aldopentose; R.S. ; Isolated


from wood; not metabolized
by humans
Glucose Aldohexose; R.S. ; Blood
sugar

- Simple sugars = carbohydrates that are quickly absorbed


by the body to produce energy Galactose Aldohexose; R.S
- Anomeric Carbon = Carbon between 2 Oxygen
- Glycosidic Bond = bond that connects the anomeric carbon
of sugar to the OH oxygen; can be either an alpha or beta Fructose Ketohexose; R.S. ;
linkage commonly found with
- Reducing Sugar (R.S.) = saccharides bearing anomeric glucose in fruit juices
carbons that have not formed glycosidic bonds
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

B. Disaccharides Glycogen
o composed of two monosaccharide units joined together by - similar in structure to amylopectin.
glycosidic bonds - stored mainly in the liver and in muscle cells
o can undergo hydrolysis - when glucose is needed for energy, glucose units are
Name Structure Description hydrolyzed from the ends of the glycogen polymer
Lactose Glu + Gal ; Milk Sugar - due to glycogens highly branched structure, there are many
; GB: -(1 -> 4) ends available for hydrolysis

Maltose Glu + Glu ; Malt Sugar Cellulose


GB: -(1 -> 4) - gives support and rigidity to wood, plant stems, and grass.
- Humans do not possess the enzyme to hydrolyze cellulose (-
glycosidase) and cannot digest it.
Sucrose Glu + Fru ; Table Sugar - makes up the insoluble fiber in our diets = important in adding
; GB: , (1 -> 2) bulk to waste -> help eliminate it more easily

Tests
C. Polysaccharides General Test
o composed of three or more monosaccharide units joined 1. Molisch Test - all 3 polysaccharides
together by glycosidic bonds 2. Anthrone Test - all 3 polysaccharides
o can be classified as: 3. Iodine Test - only glycogen & amylose
Homopolysaccharides
- consists of one type of monosaccharide Specific Test
Heteropolysaccharides I. Based on Furfural Formation
- consists of more than one type of mono. Seliwanoffs Test - for ketohexoses (FRU & SUC)
o can also undergo hydrolysis Bials Orcinol test - for pentoses (Xyloses)
II. Based on Oxidation of Sugars
Name Description Mucic Test - for GAL & LAC
Amylose linear; composed of Glu residue; found in III. Based on Reducing Property of Sugar
starch; GB: -(1 -> 4) - Makes use of the Oxidizing Agent to test the reducing property of
Cellulose unbranched; composed of Glu units; found in the carbohydrate
cell wall; GB: -(1 -> 4) - Ability of the carbohydrate to reduce metal ions like Ag+ or Cu2+
Glycogen branched; composed of Glu units; storage in alkaline medium
polysaccharide in animals; GB: -(1 -> 4) & Benedicts Test - for R.S. only (negative for SUC)
-(1 -> 6) Barfoeds Test - for Mono (negative for Disacc)
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Test Test for Reagent Principle Positive Result Other Information


-napthol in 95% EtOH -
Aldopentoses &
reacts with cyclic I. Hydrolysis of GB to
ketopentoses are
General test for polysaccharides aldehydes to form the form RS (Mono)
Molisch dehydrated to furfural
(sugars) / presence of interphase ; condensation II.Dehydration Violet Ring/ Interphase
-napthol reaction Ketohexoses are
carbohydrates reagent III. Condensation with -
dehydrated to 5-
conc. H2SO4 - napthol
hydroxymethylfurfural
dehydrating agent
I. Hydrolysis of GB to
Anthrone - condensation
General test for hexoses (diff form RS (Mono)
reagent Condensation via
Anthrone colors for diff sugars) / for sugar II. Dehydration Blue-Green Solution
conc. H2SO4 - Anthranol Intermediate
conc. III. Condensation with
dehydrating agent
Anthrone
Deep-Blue/Purple
Complexation Yellow Solution (-) =
Iodide Solution - reacts Solution (Amylose)
= the amylose in starch forms for Cellulose
General test for helical with amylose through Red-Violet Soln
Iodine helices where iodine Color Change (blue to
carbohydrates (amylose) formation of polyiodide (Amylopectin)
molecules assemble, forming a yellow or colorless) =
chains Red Solution (Glycogen)
dark blue or black color complete hydrolysis
6N HCl - dehydrating within 2 minutes
Specific test for ketohexoses (FRU agent I. Rapid Dehydration to Aldohexoses still react
Seliwanoff s Cherry-Red Solution
& SUC) Resorcinol - condensation HMF but slow and gives pink
reagent II. Condensation color
Dilute sugar soln
(0.02M) = necessary
6N HCl - dehydrating to reduce interference
agent I. Dehydration to FeCl3 = catalyst (BG
Bials Orcinol Specific test for pentoses (XYL) Orcinol - condensation Furfuraldehyde Blue-Green Solution color)
agent II. Condensation Hexoses still react but
give 5-HMF and
yields green, brown &
reddish brown colors
conc HNO3- oxidizing
Specific test for presence of Oxidation of GAL to insoluble forms a meso
Mucic agent Rhombic Crystals
galactose (GAL & LAC) Galactaric Acid (Mucic Acid) compound
Negative for Sucrose =
no free anomeric
Na2CO3 - makes
carbon
solution alkaline Oxidation of R.S. by Copper
Specific test for reducing Sodium glocunate =
Benedict s Na citrate (pH 10.5 ions to from carboxylate ion Brick-Red precipitate
carbohydrates salt of gluconic acid
in H2O) group
Green = little R.S;
CuSO4
Yellow = moderate;
Red = a lot of R.S
Rapid Oxidation of within 3 minutes
Specific test for reducing Cu acetate
Barfoed s monosaccharide in acidic Brick-Red Precipitate (Cu2O) Disaccharides will also
monosaccharides glacial Hac (pH 4.6)
conditions react but slower
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

LIPIDS
- group of structurally diverse biomolecules which are soluble in - Typed of Lipids:
organic solvent and insoluble in water Fatty acid Containing
- are not defined by a particular function groups, unlike proteins and = Waxes, Triacylglycerols, Glycerophos, Eicosanoids
carbohydrates, thus they have a variety of structures & functions Non-fatty acid lipids = Steroids
- Water insolubility due to many nonpolar C-C and C-H bonds as tails
(hydrophobic) and few polar bonds as heads (hydrophilic) FATTY ACIDS
- hydrolyzable lipids are derived from these acids
- Functions of Lipids includes the following: - are carboxylic acids (RCOOH) with long aliphatic carbon chains
Biological Membranes of 12-20 atoms
- Amphipathic Lipids = glycerophos, sphingo & cholesterol - maybe saturated or unsaturated (cis & trans)
Energy Storage = triacylglycerol - if unsaturated, can exist either monounsaturated (kink) or
Thermal Insulation & Padding polyunsaturated (essential = omega f.a.), these include:
Protective Covering = waxes Linoleic acid = omega-6
Hormones Linolenic acid = omega-3
- Polyunsaturated f.a. are needed in human diet and cannot be
- can be categorized as: synthesized by the body
- saturated = higher MP; lower BP
1. Hydrolyzable Lipids - more double bonds (unsaturated) = lower MP; higher BP
- can be converted into smaller molecules by Hydrolysis - longer chains/tail = higher MP
- includes the following: - double bonds in unsaturated f.a. are not conjugated
(1) Waxes = f.a. + long chain glycerol
(2) Triacylglycerols = 3 f.a. + glycerol COMMON FATTY ACIDS
Fats - from animals; solid/semisolid F.A. C:D.B (*) F.A C:D.B. (*)
Oils - from plants; liquid Lauric a. 12:0 -Linolenic a. 18:3 (6,9,12)
(3) Phospholipids = 2 f.a. + glycerol + phosphate Myristic a. 14:0 Arachidic a. 20:0
Palmitic a. 16:0 Gadoleic a. 20:1 (9)
2. Non-hydrolyzable Lipids Palmitoleic a. 16:1 (9) Arachidoneic a. 20:4 (5,8,11,14)
- cannot be cleaved into smaller molecules by aqueous Stearic a. 18:0 EPA 20:5 (5,8,11,14,17)
hydrolysis Oleic a. 18:1 (9) Behenic a. 22:0
- includes the following: Vaccenic a. 18:1 (11) DHA 22:6(4,7,10,13,16,19)

(1) Steroids = rings with 17C molecules Linoleic a. 18:2 (9,12) Lignoceric 24:0
(2) Fat-soluble Vitamins = Vit A, D, E, K -Linolenic a. 18:3 (9,12,15)
(3) Eicosanoids = from arachidonic acid (*) - position of double bond
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

o Waxes Experiment 6: Characterization of Saponifiable Lipids


- also called Neutral Lipids IMPORTANT SAMPLES:
- are esters formed from a fatty acid & a high molecular weight OH Glycerol
- function as protective covering for plants & animals - found in waxes, triglycerides & phosphoacylglycerols
- e.g. Spermatic wax from heads of sperm whales - simple polyol compounds (OH with multiple hydroxyl groups)
o Triacylglycerols - soluble in water
- also called Fats & Oils or Triglycerides Lecithin
- are 3 esters formed from glycerol & 3 molecules of f.a. - generic term for fatty substances in animal & plant tissues
- Nomenclature: -ic becomes -oyl + glycerol - are phosphoacylglycerols found in eggs, milk, soy bean etc.
- formed through esterification of the stated components - low solubility in water
- can be simple (3 identical f.a.) or mixed (2 or 3 diff f.a.) DCM/ Dichloromethane (CH2Cl2)
- reactions include: Hydrogenation, Hydrolysis & Oxidation - organic compound which is immiscible with water
o Phospholipids Coconut Oil
- also called Glycerophospholipids or Glycerophosphatides - extracted from kernel or meat of matured coconuts
- lipids that contain a phosphate atom - composed mainly of lauric acid
- 2 common types: - high saturated fat content
Phosphoacylglycerols - slow to oxidize, resistant to rancidification
- contain 2 f.a. + glycerol + PO4- + OH - lasts up to 2 years without spoiling
- main component of most cell membranes Canola Oil
- resemble a triglycerides with a phosphodiester bonded to an - extracted from rapeseed
alcohol replacing the 3rd f.a. - low saturated fat content
- include: Olive Oil
(1) Phosphatidylethanolamine or Cephalin (-OCH2CH2NH3) - obtained from olives
(2) Phosphatidylchloline or Lecithin (-OCH2CH2N(CH3)3) - composed mainly of oleic & palmitic acid & of other f.a.
Sphingolipids/ Sphingosine Phosphatides Palm Oil
- contain 1 f.a. + sphingosine + Phosphate + OH - derived from mesocarp of the fruit of the oil palms
- sphingomyelins have sphingosine not glycerol & amide bond - has 30 phenolic compounds
not ester as backbone & bonds, respectively - high saturated vegetable fats
o Steroids Sesame Oil
- groups of lipids whose C skeletons contain several fused rings - derived from sesame seeds
- Cholesterol - high proportion of polyunsaturated linoleic acid
= most abundant steroid synthesized in liver & found in almost all Corn Oil
body tissue; - extracted from the germ of corn
= food source: meat, cheese, butter, eggs - high smoke point
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

A. Grease-Spot Test
SV = AV + EV
- lipids, in general, have high MP & non-volatile
- Due to chemical bonds within different liquids = boils at
different temperature. C. Acrolein Test
- Oil have stronger bonds which include non-covalent - upon the sample heating with potassium bisulfate
interactions such as (dehydration), it would yield acrolein (unsaturated aldehyde)
Van der Waals forces = hydrophobic tails - further heating would result to polymerization of acrolein
Hydrogen bonding = hydrophilic heads with H2O = blackening of the reaction mixture
- Having these stronger bonds, it takes more heat to break them - test positive for phophoglycerin
apart resulting to a higher boiling point - both the pungent smell & black color indicate the presence of
glycerol
B. Saponification Test
- hydrolysis by hot alkali (3M NaOH) D. Unsaturation Test
- formation of f.a. salts = soaps - measured quantitatively in terms of the # of gram of I2
absorbed under specific condition
1. Saponification Number
= mg of KOH/NaOH needed to saponify/hydrolyze 1g of fat or oil o Iodine/Bromine Value
= also called Koettstorfer number = # of g of the halogen taken up by 100 g of fat
= gives indication of the nature of f.a. of the sample = indicates the degree of unsaturation of sample
*(the longer the C chain, the less acid is liberated per g of fat = the halogen add up to eh double bond of the lipid
hydrolyzed) = the higher # of halogen consumed, the higher # of DBs present
= inversely proportional to the MW of the molecule present = same rxn but different reactivity between the halogens
2. Acid Value (Acidity Index) - Br & I numbers cannot be compared/converted to the other
= mg of KOH/NaOH needed to neutralize the free f.a. in 1g of fat - Br is more reactive than I
= rancidity of fats is due to bacterial action
= high AV may be due to decomposition of f.a. during preparation, OIL SAPONIFICATION NUMBER
storage or purification Rapeseed/Canola Oil 170-179
= an index of the oil purity Olive Oil 185-196
Corn Oil 188-193
*(total amount of unbound f.a. are considered inversely
Palm Oil 196-205
proportional to the degree of edibility of a lipid)*
Coconut Oil 246-260
= proportional to rancidity
3. Ester Value
= mg of KOH/NaOH needed to saponify the ester of fat or oil
= indicates the adulteration of samples using paraffin or any
adulterant
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

TESTS REAGENTS TEST FOR PRINCIPLE POSITIVE RESULTS POSITIVE IN


Grease Spot Test H2O, DCM, Lecithin, Presence of lipids Determination of Translucent grease All Vegetable Oil
Oil high boiling point marks Sample & Lecithin
Saponification Test 3M NaOH Presence of ester Hydrolysis of ester Presence of Bubbles Oil & Melted Fat
(hydrolyzing agent), bonds linkages under basic & Precipitate = Soap
H2SO4 conditions to form
(neutralizing agent) glycerol & f.a. salts
Acrolein Test KHSO4 Presence of glycerol Oxidation & Blackening of rxn Phosphoglycerin
(dehydrating agent) dehydration with the mixture & pungent (Lecithin)
presence of heat smell
Unsaturation Test DCM = used to Presence of double Halogenation of Colorization of Oil, Glycerol, Melted
dissolve lipids & bonds & degree of alkenes via Solution Fat
make the sample unsaturation bromination (Brown Orange
easily with 5% Br2- Solution due to Least Unsaturated:
DCM Excess Bromine) Coconut Oil
5% Br2-DCM
(halogenizing agent) Most Unsaturated:
*why DCM? Sesame Oil
(1) good solvent for
Br & (2) doesnt
react with Br

EXERCISE

Sample AV SN IN EV
A 9 215 68 206 Best Keeping Quality: Iodine Number
B 90 250 46 160 Lowest MW: SN of B
C 50 190 96 140 Least Susceptible to Oxidation: IN of B
Greatest # of Ester bonds: EV of A
SV = AV + EV Least tendency to melt at room temp: IN of C
Least tendency to become rancid: AV o
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Experiment 7: Isolation & Characterization of Complex Lipids ISOLATION OF LIPIDS FROM CALF BRAIN

Simple Lipids
- esters of f.a. with OH
- include fats, oil and waxes

Complex Lipids
- esters of f.a. containing groups in addition to an OH & a
f.a
- include glycolipids like cerebroside and phospholipids

CERERBROSIDE
= ceramide + monosaccharide (D-glu or D-gal)
= can occur as glucocerebroside or galacerebroside
* Accomplished by successive extraction with various elements
PHOSPHOLIPIDS
o Acetone
= contain phosphoric acid (H3PO4)
- provides a mild but rapid method of dehyrdrating tissue
= include (1) Lecithin, (2) Cephalin, (3) Phosphatidyl
- as H2O content decreases, it extracts fats, sterols, & other lipids
Serine & (4) Sphingomyelin
- complex lipids are relatively insoluble in it, thus it is extracted
with other solvents
Phosphorylated Lipids (PL)
- easy to remove by evaporation after being used as a solvent
- have polar & nonpolar groups (amphipathic)
- separates the phosphorylated lipids from non-phosphorylated
- contain phosphoester groups
lipids (NPL)
- lipids that contain other groups of atom other than C,H,O
- NPL (e.g. sterols) will dissolve in acetone because they are both
PHOSPHATIDYLCHOLINE (Lecithin)
highly nonpolar molecules
= phosphatides of choline
TESTS C G S
= glycerophospholipid
L-B YES NO NO
Salkowski YES NO NO
Non-phosphorylated Lipids (NPL) Krauts NO YES YES
- no other constituents other than C,H,O Ninhydrin NO YES YES
TRIGLYCERIDES (Fats & Oils) Phosphate NO YES YES
= 3 f.a. esterified to glycerol Molisch NO NO YES
= neutral & non-polar Acrolein NO YES NO
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

TESTS REAGENTS TEST FOR PRINCIPLE POSITIVE POSITIVE IN


RESULT
Condensation of Acetic
Liebermann- Acetic Anhydride General Test for Emerald-Green
Anhydride with the C-3 - Cholesterol, NPL
Burchard Test conc. H2SO4 Sterols Solution
OH & C-5 double bond
CHCl3
conc. H2SO4 Condensation of 2
Specific Test for Red to Purple
Salkowskis Test = react with Cholesterol cholesterol molecules to Cholesterol, NPL
Cholesterol Interphase
resulting to red from bisteroids
biocholestadien disulphoate
Krauts Reagent (KBiI4) = Specific Test for Complexation Reaction of
Brick-Red Glycerophosphatides,
Krauts Test potassium iodide + bismuth Presence of Choline Choline with KBiI4
Precipitate Sphingolipids, PL
subnitrate (Lecithin) (formation of colored salt)
General Test for
Ninhydrin Presence of Amino
(1) Oxidative Deamination Glycerophosphatides,
Ninhydrin Test (triketohydrindene hydrate) Acids (Free Amino Violet Solution
(2) Condensation Sphingolipids, PL
in EtOH group & Secondary
Amines)
Fusion Mixture
(KNO3/Na2CO3, 3:1)
= oxidizing agent General Test for
Phosphate (or = use to convert organic Presence of Yellow Glycerophosphatides,
Oxidation Reaction
Nitrogen) Test compounds into inorganic Phosphate (or Precipitate Sphingolipids, PL
form Nitrogen)
3M HNO3, 2.5%
ammonium molybdate
(1) Hydrolysis of glycosidic
bond forming the reduced
-napthol in 95% EtOH General Test for sugar (mono)
Molisch Test Sphingolipids,
= condensation reagent Presence of Sugar (2) Dehydration of the Violet Ring/
(-napthol Galactocerebrosides,
conc. H2SO4 Moiety mono into hydroxymethyl Interphase
reaction) NPL
= dehydrating agent (Carbohydrates) furfurals
(3) Condensation of the
furfural with -napthol
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

NUCLEIC ACIDS
- molecules that store info. for cellular growth & reproduction 2. Pentose Sugar
- include deoxyribonucleic acid (DNA) & ribonucleic acid (RNA) - has C atoms numbered with primes to distinguish them
from the atoms in N.B.
NUCLEOTIDES - RNA is ribose
- monomeric building blocks of Nucleic Acids - DNA is deoxyribose (no O atom on carbon 2)
- Nucleosides are nucleotides without phosphate
- Linkages include:
(1) -N-glycosidic bond = links N.B. to P.S.
(2) Phosphoester bond = links P.S. to phosphate

2
1 3. Phosphate
- gives nucleic acids its negative charge
- makes nucleic acid attracted to ANODE pole
- Nucleotides contain the following:
1. Nitrogenous Base NucleoSide NucleoTide
a) Pyrimidines - has a base linked by a - is a nucleoside that forms
- heterocyclic 6 membered aromatic ring glycosidic bond to C1 of a a phosphate ester with the
- include C & T for DNA and C & U for DNA sugar C5 OH group of a sugar
- named by changing the - named using the name of
the base ending to: the nucleoside +
-idine for pyrimidines 5-monophosphate
-osine for purines

b) Purines
- 2 heterocyclic fused aromatics
- include A & G for both DNA and RNA
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Homogenizing Solutions
1. Sodium dodecyl sulfate (SDS)
- anionic detergent which disrupt ionic interactions between
proteins that holds membranes together
- breaks down nuclear and cell membrane
2. NaCl Solution (Saline Soln)
- increases the solubility and stability of DNA
- provides sodium ions that neutralize the negative charge of
the DNA backbone (phosphates)
3. Sodium citrate
- inactivate DNAse by trapping Mg2+and Ca2+which acts as
cofactor of the enzyme
4. Ethylenediaminetetraacetic (EDTA)
RNA DNA - binds divalent metal ions (Cd2+,Mg2+, Mn2+ that could form
Sugar Ribose Deoxyribose salt with anionic PO43-group of DNA
N.B. A,U,G,C A,T,G,C
Strand(s) Single Double II. Deproteinization
Function transfer & expression of storage of genetic - purify the homogenate by removing proteins that are
genetic information information associated with cells & DNA molecules
- dissociation of protein-DNA complex by denaturation
Experiment 8: Isolation and Characterization of DNA from - Papain
Allium cepa (Onion) = degrades DNAases, RNAases, and proteins
Why White Onion? = proteolytic enzyme that denatures the proteins attached to
- Onion = low starch content allows DNA to be seen clearer DNA making it flexible & easy to spool
- White = longer strands of DNA compared to red onions - Why transfer the flask immediately into an ice bath?
I. Homogenization = slows down DNA breakdown
- breaking up of onion tissues to separate & open cells = at room temp, DNA begins to denature by Dnases action
- release of DNA in soluble form by destruction of cell - Why is the onion tissue mixed in a blender for 45 sec
membranes and membranes of subcellular particles (nuclei) only?
- Involves heating and blending the onion tissues in order to = to allow DNA release with homogenization media and
break down the cell breaks down cell wall and membrane
- Heating at 60C softens the phospholipid in the cell = exceeding to 45 sec exposes the DNA to shear forces that
membrane and denatures DNAse (higher temp = thermal rapidly break the DNA into shorter lengths
denaturation)
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

III. DNA Precipitation Acid Hydrolysis


- force DNA to come out of soln or ppt, by using cold OH - Why not Alkaline Hydrolysis?
- separation of DNA from other biomolecule = pentose sugar of DNA does not have an OH at 2 C
= no formation of monophosphate intermediate
- Why use cheesecloth for filtering homogenate? = DNA is stable with alkaline hydrolysis
= to remove bigger particles such as cell wall, membranes and
any solid material - Reagents:
= more efficient compared to filter paper resulting in a clear cell 1.0M HCl
homogenate = dissociates DNA into its components
= caused depurination
- Why use ice-cold 95% EtOH? 1.0M NaOH
= precipitates out DNA in the form of fibers = neutralizes the soln so it wont affect the color test
= 95% EtOH is not soluble with DNA and decreasing temp will
result to lower solubility to other particles - Why is the DNA-HCl mixture heated to 100C?
= having an lower dielectric constant than water, EtOH allows = to destroy hydrogen, phosphoester and glycosidic bonds
sodium ions from saline soln to interact with the negatively = DNA components are dissociated into (1) Phosphate
charged phosphate groups closely enough to neutralize them & group, (2) Purine & Pyrimidine & (3) Deoxyribose
let DNA fall out of soln
- Results (breaking of bonds associated to DNA components)
UV-Vis Analysis o Hydrogen Bond
- determines the purity of the isolated DNA = links the base pairs (3 for C&G; 2 for A&T)
- Absorbance measurements at 260 nm (DNA) 280 nm = heating the temp above 80C
(protein) o Phosphoester Bond
- A260/280(Absorbance ratio) = links the phosphate & pentose sugar
- Ideal ratio ranges from 1.7 to 1.8 = heating the temp above 90C
< 1.7 = high amount of protein = acidic with pH less than 2
1.8 = high amount of DNA o (-N-) Glycosidic Bond
- Using a nomograph, the amount of protein and nucleic acid = links the nitrogenous base & pentose sugar
can be determined = acidic with pH less than 2
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

TESTS STANDARD REAGENTS PRINCIPLE POSITIVE RESULT


1. Dehydration of deoxyribose forming
Deoxyribose conc. H2SO4,
Dische Test hydroxylevulinaldehyde
standard (Dehydarating Agent) Blue Solution
(for Deoxyribose) 2. Complexation reaction with
solution Diphenylamine reagent
Diphenylamine
1. Oxidation of Purines forming dialuric
acid & alloxan
conc. HNO3,
Murexide Test Solid 2. Condensation reaction forming
(Oxidizing Agent) Purple Red Residue
(for Purines) Guanine alloxanthine
10% KOH
3. Neutalization producing the purple red
murexide/ ammonium purpurate
1. Bromination of Pyrimidine to form
Wheeler-Johnson
Cytosine Bromine Water, Dialuric Acid
Test Purple Solution
solution Ba(OH)2 2. Neutraliztion of D.A. to from Barium
(for Pyrimidines)
salt of D.A.
Phosphate conc. HNO3, Yellow Precipitate
Phosphate Test Precipitation of Phosphate
solution 2.5% NH4MoO4 solution (NH4)3PO412MoO3

Experiment 9: Urinalysis
Initial Examination of Urine Samples
Urine
- by-product secreted by kidney after removing soluble waste 1. Color
products from the body - color comes primarily from the presence of Urobilin
- reflects the work of kidneys to maintain homeostasis - Urobilin = final waste product resulting from the
- transported by the ureter to the urinary bladder & voided breakdown of heme from hemoglobin during the
through urethra destruction of blood cells
- Urinalysis, used since the time of Hippocrates (the father of - Normal: Clear Yellow to Yellow Orange
medicine), detects abnormally high amount of normal & Hematuria blood in urine
pathologic constituents which indicates a disorder or Melanuria black or dark urine caused by
malfunctioning metabolic reaction melanoma
- Urine Composition includes: Porphyria reddish or brown
o 95% Water and other constituents (Sulfate, Phosphate, Jaundice or Gilberts Syndrome dark orange or
Chloride, Magnesium, Calcium, Potassium, Sodium, dark colored
Ammonia, Creatinine, Uric Acid)
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

2. pH Biochemical & Medical Uses:


- urine pH is usually acidic with pH of 6.6 (1) Diagnostic Use and Drug Tests
- 24 hr samples are less acidic due to decomposition of urea (2) Method for excreting toxins, chemicals & drugs from
to ammonia the body
- Tested using (1) pH paper or (2) litmus paper
- Normal: pH range of 4.8-7.5 Industrial Uses:
Hyperuricosuia acidic urine which contribute to (1) Fertilizer (1:5 with water)
uric acid stone formation in kidney, ureter & (2) Gunpowder (Nitrogen)
bladder (3) Cleaning Fluid (Ammonia)
(4) Teeth Whitening used in Ancient Rome
3. Turbidity
- Normal: Clear & Transparent Organs of the Urinary System: Kidney, Ureter, U.B., Urethra
Turbid / Cloudy Urine symptoms of bacterial
infection and is caused by crystallization of salts Urination
(calcium phosphate) - voluntary control of the release of urine from the U.B.
through the urethra to the outside of the body
4. Odor - Urination of infants and people with neurological injury is
- reflects of what has been consumed or specific diseases involuntary
- Normal: aromatic odor
Putrid/Strong Ammoniacal decomposition of urea Urea Cycle
to ammonia - cycle of biochemical reactions occurring in some
Fruity Aroma diabetic patients due to large organisms the produces urea ((NH2)2CO) from ammonia
amount of acetone & ketone bodies (NH3)
- also known as ornithine cycle and the first metabolic cycle
5. Volume discovered by Hans Krebs & Kurt Henseleit in 1932
- depending on the state of hydration, activity level, - Urea cycle takes place primarily in the liver
environmental factors, weight & individuals health Step 1 (in mitochondria)
- Normal: 1-2 L/day - ammonia from amide nitrogen of Glutamate (1st AA)
Polyuria excessive production (>2.5L/ day) - carbamoyl phosphate enters the cycle as a donor
Oliguria - < 400 mL/ day Step 2
Anuria - < 100 mL/ day - C. P. donates its Carbamoyl group to Ornithine to form Citrulline
Step 3
- Aspartate (2nd AA) + Carbonyl of Citrulline = Argininosuccinate
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Step 4 Qualitative Examination for Normal Organic


- only reversible step in the cycle Constituents
- Fumarate enters mitochondria to participate in Krebs Cycle (for readily/ always present substances in every urine sample)
- Cytosolic enzyme arginase cleaves arginine to yield urea & orni A. Test for Urea
- Ornithine enters the mitochondria Ammonia
- = extremely toxic base & accumulation would quickly be fatal
How is Urine formed? (in Nephrons) = together with CO2, converted by liver into urea through urea cycle
1. Filtration = though the body cant tolerate high conc, urea is less poisonous
- blood pressure forces small molecules from the glomerulus than ammonia and efficiently removed by kidneys
to the capsule B. Test for Uric Acid
- Filtrates: glucose, amino acids, uric acid & urea Uric Acid
- = product of nucleic acids metabolism
2. Reabsorption = produced within peroxisomes & slightly soluble in H2O
- Water Reabsorption: return of H2O via osmosis along the = easily precipitates out of soln forming needle like crystals of
loop of Henle & collecting ducts sodium urate -> formation of kidney stones & pain in gout when
- return of filtrates from blood at the proximal tubule through deposited in joints
diffusion and active transport = kidneys reclaim most of the u.a. filtered at the glomeruli
- 2 Step Process: C. Indican Test
(1) Extraction of Substances from the Tubule Fluid into the Indican
Renal Interstitium = product of converted and absorbed indole from intestinal bacterial
(2) Transport of substances from the interstitium to the cleavage of tryptophan
blood stream = also known as 3-hydroxy indole or indoxyl
= transported through blood to the kidneys for excretion
3. Tubular Secretion = metabolized indoles turn into skatole that produce indicans like
- movement of molecules from blood into the distal indoxyl potassium sulfate & indoxyl glucoronate -> bacterial activity
convoluted tubules in the small & large intestines
- peritubular capillaries to the renal tubular lumen = elevated levels are indicator of intestinal toxemia & overgrowth of
- Secreted molecules: drugs, excess water and toxins anaerobic bacteria
D. Test for Creatinine
Time of Collection: First in the Morning Creatinine
= higher concentration of constituents = protein produced by the muscle & released into the blood
= has dark to light yellow color = creatinine level in the serum determined by the rate of its removal
= more likely to yield abnormalities if present roughly measures kidney function
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

Qualitative Examination for Pathologic Organic


Constituents

A. Gunnings Test (for Ketone Bodies) C. Extons Test (for Albumin)


- exist in urine in the form or acetone, acetoacetic acid, - Albumin
hydroxybutyric acid = proteins like albumin are not normally found in urine but
Ketone Bodies should be keep in blood by the kidney
= chemical product of the body when insulin is not enough = normally reabsorbed and use as sources of energy
in blood & it must breakdown fat = if kidneys is diseased, protein will appear in urine even if
= can poison & kill body cells blood levels are normal or not
= without insulin, ketones build up in the blood & spill
over into the urine to be excreted by the body D. Smiths Test (for Bile Pigments)
Bilirubin
B. Benedicts Test (for Glucose) = product of hemoglobin breakdown
Glucose = conjugated to acid in liver
= end product in blood of most dietary carbohydrates = unconjugated ones are water soluble & excreted in urine
= required for brain to function normally = abnormal levels may indicate anemia, excessive RBC
= high glu conc (>300mg/dl) cause similar symptoms if breakdown, hepatitis, cirrhosis, obstruction of biliary duct
associated with dehydration, infection or acidosis & tree and toxic level damage
= levels of it are measured to diagnose or control diabetes
= Diabetes results from deficient or decreased sensitivity to E. Test for Occult Blood (for RBC in Urine)
insulin Blood Urine
= urine glucose test are abnormal in cases of renal = from a problem in kidneys or other parts of the U. Tract
glycosuria & diabetes mellitus = these problems include the following:
Kidney & Bladder Stones
Infection of Kidney, Bladder (chronic or recurrent)
& Urethra (tube that empties urine from bladder)
Inflammation of Infection of Kidney
(glomerulonephritis), Bladder & Urethra
Cancer of Kidney or Bladder
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

TESTS REAGENTS PRINCIPLE POSITIVE RESULTS OTHER INFORMATION


- from ammonia (urea
70% NaOH, Production of N2 gas when cycle)
Test for Urea Evolution of N2 gas
Bromine Water urea react with NaOBr - removed efficiently by
kidneys
- product of nucleic acid
metabolism
20% Na2CO3, Redox of Uric acid by Formation of Blue -Disease: Kidney Stones &
Test for Uric Acid
Phosphotungstic Acid P.Acid Solution Gout (forms sodium urate
crystals with water)
- filtered in glomeruli
-Disease: Intestinal
Toxemia & Overgrowth of
Obermayers Reagent, Oxidation of Indoxyl by Formation of Blue color in
Indican Test Anaerobic Bacteria
Chloroform Ferric Ions the Bottom Layer
- Tryptophan >Indole
>Indican
- produced by the muscle
Alkaline Picrate Solution Rxn of P.A.in alkaline
Formation of Orange - level in serum determines
Test for Creatinine (5:1 saturated picric acid- medium reacts with
Colored Solution the rate it is removed
10% NaOH) creatinine
shows the kidneys func
BIOCHEMISTRY LAB ALVIAR [3BIO1; 2017]

TESTS REAGENTS PRINCIPLE POSITIVE RESULT OTHER INFORMATION


conc. ammonium
hydroxide
Gunnings Test (for Lugol Solution Rxn of acetone to form Formation of Iodoform - exist in urine in form of
Ketone Bodies) KI - form iodo crystals iodoform crystals Crystals acetone, acetoacetic acid
Ammonium hydroxide -
basifies the sample
Benedicts Reagent
Reduction of Copper
Na Citrate - Buffer - Diseases: Diabetes mellitus
Benedicts Test (for Sulfate to Cuprous Oxide Yellow to Red
Na Carbonate - Alkali (deficient/ dec sensitivity to
Glucose) in the presence of heat & Precipitate
Copper Sulfate - Reduc alkali
insulin) & Renal Glycosuria
& Ppt Agent
Extons Reagent
Anhydrous Na Sulfate - Acid Precipitation of
Extons Test (for White Precipitate with
Drying Agent Albumin with Heat by - from Kidney
Albumin) Cloudiness
Sulfosalicylic Acid- Ppt Sulfosalicylic Acid
Agent
- bilirubin from hemoglobin
breakdown
- unconjugated bilir. are
Tincture of Alcoholic soluble with H2O
Bilirubin, Bilicyanine & Formation of Emerald
Smiths Test (for Bile Iodine Mixture (Iodine - Diseases: Anemia,
Choletellin - depend on Green Interphase /
Pigments) Crystals & Sodium Iodide Hepatitis, Cirrhosis,
Oxidation of Bile Pigments Point of Contact
in Absolute EtOH) excessive breakdown of
RBC, Obstruction of Biliary
Duct & Tree, Toxic Liver
Damage
Peroxidase Activity of - Diseases: Kidney Stones;
95% EtOH with Guaiac
Hemog. which decomposes Bladder, Kidney & Urethra
Powder,
Test for Occult Blood H.Perox & liberated Inflammation & Infection;
Hydrogen Peroxide, Blue Ring
(for RBC in Urine) Oxygen that oxidizes Cancer of Kidney & Bladder
Glacial Acetic Acid -
Benzidine & Guaiac *Glomerulonephitis =
adifies
Powder Kidney Inflammation