Lessons 1-2.

Water, fluid compartments of

the body, internal

environment, homeostasis,

isovolaemia, isosmosis,

isoionia, isohydria (Susan

Neogrady)

Information, announcements, topics

list, downloading theoretical lessons

and lab manualHomepage of Biochemistry:

https://univet.hu/en/biochemvet/

General information

• Veterinary Biochemistry is taught during two semesters,

in the following theoretical and practical class time:

• Biochemistry 1.: 45 hours lectures

• practicals (5x3 hours) 15 hours

• Biochemistry 2.: 45 hours lectures

• practicals (5x3 hours) 15 hours

• General consultation: orban.kata@univet.hu

Theoretical lectures

• Introduction. Internal environment, homeostasis: isovolaemia, isotonia, isoionia and isohydria. Buffer systems. Structure of the biological membranes.Transports across the biological membranes. General characteristics of proteinsClassification and structure of proteinogenic amino acids. Peptide bond. Structure of proteins. Denaturation and renaturation of proteins.cClassification of proteins. Collagen, elastin, keratin.

• Characterization of enzymes. Mechanism of enzyme action. Reversibility of enzymatic reactions. Factors influencing the velocity of enzymatic reactions. Regulation of enzyme activities. Zymogens, isoenzymes.Nomenclature, classification and cellular localization of enzymes.

Theoretical lectures• Molecular biology: Structure of nucleotides. De novo

synthesis and degradation of purine nucleotides. De novosynthesis and degradation of pyrimidine nucleotides. Salvage pathways of nucleotide metabolism. Synthesis and degradation of deoxyribonucleotides. Structure and function of DNA. Replication of DNA in prokaryotes and eukaryotes. Mutations, repair mechanisms. Transcriptionand its regulation in prokaryotes and eucaryotes.

Transcription and its regulation in procaryotes and eukaryotes. Influencing of gene expression. Epigenetic regulatory mechanisms. Translation. Structure of the ribosomes. Activation of amino acids. Initiation of the translation. Elongation and termination of the translation. Posttranslational modifications and transport of proteins.Recombinant DNA technology and biotechnology.

Midterm

• The midterm is accepted if the student completes 60%

(18 points) of the maximal scores (30 points). If the

midterm is failed, there is a second possibility to retake it.

• The curriculum and requirements of the midterm retake

are the same as the first midterm. Tests are on display in

a limited interval.

• If the midterm retake is failed, there is an extra possibility

to get the semester accepted: a written end-term

examination has to be taken from each topic of the

semester. The result of the examination is scored and can

be accepted if min. 18 points (60%) has been reached.

Requirements of the acceptance of

Biochemistry 1.• Fulfilling the requirements of the practical part of Biochemistry 1.:

• Successful performance of all laboratory courses.

• Completion of 60% (18 points) of the maximal scores (30 points) in

the laboratory courses. If the written midterm test (or the midterm

retake or the end-term examination=midterm re-retake) has not

been completed, but the requirements of the practical part of

Biochemistry 1. have been fulfilled, the semester is not accepted;

however, only the midterm has to be repeated during a later

semester. But please note that Biochemistry 1. labs will be held only

in the fall semester of each academic year.

51 – 55 points: excellent (5)

45 – 50 points: good (4)

40 – 44 points: medium (3)

33 – 39 points: satisfactory (2)

Laboratory practical courses in Biochemistry

• Qualification system

• In the labs the students have to give an account of the principle and the practical essence of the different experiments. The evaluation of the knowledge of the students consists of two parts:

• 1. Short written test at the beginning of the lab, regarding the theoretical and practical essence of the experiments and the appropriate theoretical subject-material. At certain labs, the appropriate chemical structures, in connection with the lab topic, will be also asked in the test. The structure list can be downloaded from the webpage of the Department.

• 2. Students will be asked at the end of the lab concerning their results

Order of the laboratory courses in the

first „Biochemistry” semester

• Topics of the practicals Biochemistry 1.:

• 1. Examination of some biochemical parameters of the blood plasma

• 2. Examination of proteins

• 3. General properties of the enzymes, enzyme activity

• 4. Examinations of enzymes of digestion

• 5. Examination of nucleic acids

Essential readings

• 1. Your own Lecture Notes written during the lectures

2. Downloads (Homepage of the Department of Physiology and Biochemistry): Lecture Notes by Susan Neogrady

• Biochemistry, Laboratory Manual: Downloads: Homepage of the Department of Physiology and Biochemistry

•3. University TextbooksIntroduction to the Intermediary Metabolism (Prof. Ferenc Kutas)

• The Molecular Basis of Biochemistry (Prof. Ferenc Kutas)

• Biochemistry of Vitamins (Prof. Ferenc Kutas)

Recommended readings

• Harper's Illustrated Biochemistry (LANGE Basic Science) by Robert K. Murray, Darryl K. Granner, Peter A. Mayes, and Victor W. Rodwell (Paperback - Mar 18, 2003)

• Biochemistry (with BiochemistryNow) by Mary K. Campbell and Shawn O. Farrell (Hardcover - Jan 14, 2005

• Biochemistry (with Lecture Notebook) by Mary K. Campbell and Shawn O. Farrell (Hardcover - Oct 29, 2002)

• Lehninger Principles of Biochemistry (Albert L. Lehninger, Hardcover)

• Lehninger Principles of Biochemistry 4e + Cd-rom (David L. Nelson Ph.D., Hardcover)

• Lehninger Principles of Biochemistry, Fourth Edition + Lecture Notebook (David L. Nelson Ph.D., Hardcover)

• Biochemistry, Fifth Edition: International Version by Jeremy M. Berg, John L. Tymoczko, and Lubert Stryer (Paperback - Feb 15, 2002

• Biochemistry by Jeremy M. Berg, John L. Tymoczko, and Lubert Stryer (Hardcover -May 19, 2006)

• BRS Biochemistry and Molecular Biology (Board Review Series) (Paperback) by Todd A Swanson (Author), Sandra I Kim (Author), Marc J Glucksman (Author)

•

During the lectures:

http://msmeans.wordpress.com/2012/05/07/back-in-the-saddle-keeping-life-balance-despite-the-challenges/

BALANCE: CONSTANCY OF

THE INTERNAL ENVIRONMENT

HOMEOSTASIS

WATER (60% OF THE BODY)

• General characteristics

• Water is often regarded as a bland, inert liquid, a space filler in the organism. It is not right. Water is a highly reactive substance with unusual properties.

• Water is a very polar molecule due to the large difference in the electronegativity (tendency of an atom to attract electrons) of oxygen (3.5) and hydrogen (2.1). In the water molecule the hydrogens and oxygen are covalently bonded to each other, but because of its polar character, one side of the molecule has a partial positive charge, while the other has a partial negative charge.

• Polar molecules (dipoles) interact well with ions and other dipoles.

WATER MOLECULE

• (by Martin Chaplin )

WATER AS SOLVENT

Ionic and polar neutral substances tend to

dissolve readily in water. These

substances are called hydrophilic (water

loving), water soluble compounds.

Which are non-polar are hydrophobic

(water fearing) molecules, that means,

they are non-water soluble (lipophilic).

Water detemines the structure and

biological properties of

• Proteins • http://www.chemguide.co.uk/organicprops/aminoacids/proteinstruct.html

• Nucleic acids• http://www.3dscience.com/3D_Models/Biology/DNA/DNA_with_Phosphate.php

• Lipids• http://images.tutorvista.com/content/cellular-micromolecules/lipid-bilayer-structure.jpeg

• Biological membrane• http://www.cytochemistry.net/cell-biology/membrane_intro.htm

WATER AND pH

• Water has a tendency to ionize into hydrogen and hydroxyde ions. This ionization is crucial to water’s role in cellular function. The amount of hydrogen and hydroxide ion in a solution is always related by the equation:

• [H+] x [OH-] = 10 -14 mol/l

• In pure water, there is one hydrogen ion for every hydroxide ion, so they are both equal 10 -7 mol/l

• As pH = -log [H+], in pure water pH = 7

• Water and its ionization products are important factors in determining the structure and biological properties of proteins, nucleic acids lipids, membranes etc.

• The water content of the living organisms are relatively high.

INTERNAL ENVIRONMENT

• As Claude Bernard repeatedly stated, life

is an expression of the physical reality and

the maintenance of life is guaranteed by

the constancy of the fluid matrix or

‘milieux interieur’ (Bernard).

CLAUDE BERNARD (1813-1878)

A FRENCH PHYSIOLOGIST

•

• Memorial plaque in Paris marking the site

of Claude Bernard's laboratory from 1847

until his death in 1878.

Stability of internal environment

=HOMEOSTASIS

• Walter Bradford

Cannon

• Homeostasis

• from "The Wisdom of

the Body" (1932)

Walter Cannon

(1871-1945)

Stability of the internal

environment=HOMEOSTASIS

http://www.harvardsquarelibrary.org/unitarians/cannon_walter.html

The constant conditions which are maintained in the body

might be termed equilibria. That word, however, has come to

have fairly exact meaning as applied to relatively simple physico-

chemical states, in closed systems, where known forces are

balanced. The coordinated physiological processes which maintain

most of the steady states in the organism are so complex and so

peculiar to living beings - involving, as they may, the brain and

nerves, the heart, lungs, kidneys and spleen, all working

cooperatively - that I have suggested a special designation for

these states, homeostasis. The word does not imply something

set and immobile, a stagnation. It means a condition - a condition

which may vary, but which is relatively constant. "The Wisdom

of the Body" (1932)

Fluid compartments in the body(dog, body mass: 20 kg)

Total body fluid

12kg (60% of the body

mass)

Intracellular fluid (ICF)

2/3

8kg (40% of the body mass)

Extracellular fluid (ECF)

1/3

4kg (20% of the body mass)

Interstitial fluid (ISF)

3/4

3kg (15% of the body mass)

Intravasal fluid (IVF)

1/4

1kg (5% of the body mass)

Transcellular fluid (TCF)

~1%

~0.1kg

http://www.magyar-vizsla.eoldal.hu/fenykepek/magyar-vizsla/magyar-20vizsla-1-.html

FLUID COMPARTMENTS IN

THE BODY• Intracellular Fluid (ICF) comprises 2/3

of the body's water. If the body has 60%water, ICF is about 40% of the body mass.The ICF is primarily a solution of potassium and organic anions, proteins etc.The cell membranes and cellular metabolism control the constituents of this ICF. ICF is not homogeneous in the body. It represents a conglomeration of fluids from all the different cells.

FLUID COMPARTMENTS IN

THE BODY• Extracellular Fluid (ECF) is the remaining 1/3 of the

body's water. ECF is about 20% of the body mass.The ECF is primarily a NaCl and NaHCO3 solution.The ECF is further subdivided into three subcompartments: Interstitial Fluid (ISF) surrounds the cells, but does not circulate. It comprises about 3/4 of the ECF (15% of the body mass). Blood plasma (intravasal fluid: IVS)circulates as the extracellular component of blood. It makes up about 1/4 of the ECF (5% of the body mass).The 3rd subcompartment is transcellular fluid (TCF) is a set of fluids that are outside of the normal compartments. These 1-2 % of fluid make up the cerebrospinal fluid in the CNS, aqueous humor and vitreous humor in the eye, synovial fluid in the joints, glandular secretions, and serous fluid etc.

60% of body mass: FLUID

COMPARTMENTS IN THE BODY (www.auburn.edu)

HOMEOSTASIS, STABILITY OF

ECF

• Components:

• 1. Isovolemia

• 2. Isosmosis

• 3. Isoionia

• 4. Isohydria

ISOVOLAEMIA: THE

CONSTANCY OF THE VOLUME

OF ECF• Dehydration=decreased volume of ECF = less than than 20%

• (no drinking water, diarrhea, vomiting, increased sweating, etc)

• Overhydration= increased volume of ECF = more than than 20%

• (renal diseases, too much drinking, too much infusion, etc)

• Hypovolaemia = decreased volume of blood plasma

• (bleeding, shock, etc)

• Hypervolaemia = increased volume of blood plasma

• (cardiac, renal diseases, etc)

• Oedema: increased volume of ISF

• (cardiac decompensation, hepatic, renal diseases, starvation, allergic processes, inflammation, etc)

Vomiting, diarrhea, sweating few

drinking water lead to dehydration

Bleeding results in hypovolaemia

OEDEMA: increased volume of

interstitial fluid• (Mediscan)

Severe oedema of the comb, wattles

and periorbital area, Oedema in cattle,

swelling of the face

ISOSMOSIS: THE CONSTANCY OF THE

OSMOTIC PRESSURE (the force which

prevents the osmotic movement) OF ECF• Osmotic pressure depends on the number of

solute particles per unit volume=osmotic concentration (osmolarity)

• 1 mol glucose = 1 osmol1mol NaCl = 2 osmol (Na+, Cl-)1mol CaCl2 = 3 osmol (Ca2+, 2xCl-)

Osmotic movement http://www.google.hu/imgres?q=osmotic+movement&um=1&hl=hu&biw=1061&bih=392&tbm=isch&tbnid=Wr044lXwQQF3rM:&imgrefurl=http://www.bbc.co.uk/

bang/handson/rubber_egg.shtml&docid=YvQneNB-

G_KPAM&imgurl=http://www.bbc.co.uk/bang/images/446x251/osmosis.jpg&w=446&h=251&ei=cHgjUrGyOMi0tAa4jIDoCw&zoom=1&iact=hc&vpx=2&vpy=99&

dur=3422&hovh=168&hovw=299&tx=161&ty=109&page=1&tbnh=132&tbnw=226&start=0&ndsp=10&ved=1t:429,r:5,s:0,i:94

C1>C2 C1=C2

Determination of the osmotic

concentration=osmolarity

• Determination of osmotic concentration of the

blood plasma by measuring freezing point

depression. 1 osmol solute dissolved in 1 l

solution depresses the freezing point by 1.86oC.

Measuring the freezing point depression of the

blood plasma the result is -0.56oC . To calculate

it: proportionally means (1.86:0.56=1:X) about

0.3 osmol (=300 mosmol) solute per liter

solution. 0.9%NaCl!!!!!!! (physiological

saline)=0.3 osmol

Red blood cells: living osmometers

• In isotonic solution: no water movement across the membrane

3% NaCl

RBC shrinks

0.9% NaCl, in the

NaCl solution:

isosmotic=isotonic

Shape of RBC

does not change

0.3% NaCl

RBC disrupts

(hemolysis)

Isosmotic or isotonic?

• For an isosmotic solution to be isotonic, the

membrane must be equally impermeable to all

solutes.

• All isotonic solutions are isosmotic!

• But not all isosmotic solutions are isotonic!!

• But in the everyday clinical praxis isosmotic and

isotonic are used as synonimes.

Red blood cells in hypertonic

solution

Red blood cells in isotonic solution

Red blood cells in hypotonic

solution

0.025M

Sucrose

Red blood cells in hypotonic solution will

swell and disrupt (like the cherry in the rain)

Izoionia

ECF (mmol/l) ICF (mmol/l)

Cation Na+ 140 27

K+ 5 95

Ca2+ 2.5 (total) 1

Mg2+ 1 3

Anion Cl- 103 30

HCO3- 27 10

H2PO4-

/HPO42-

1.5 30

Pr- 16 60

ISOHYDRIA: THE CONSTANCY

OF THE pH OF ECF• pH of ECF: 7.4

• In generally acids could be called as H+

(proton) donors and bases as H+ (proton) acceptors. If an acid is weak, it has high affinity for protons and dissociates slightly. If an acid is strong, it has low affinity for protons and dissociates intensively.

• The tendency of any given acid to dissociate (is a weak or strong acid) is given by its dissociation constant of the acid (Ka).

HENDERSON-HASSELBALCH

EQUATION

• Ka=[H+]x[A-]/[HA]

• The negative logarithm of Ka is pKa (and

the negative logarithm of [H+] is pH), so we

gain the next equation.

• pH=pKa+log[A-]/[HA] Henderson-

Hasselbalch equation,

• weak acids have high pKa values, strong

acids have low pKa values.

pKa VALUES (example for better

understanding)

• The pKa values are not magic numbers, it

can be determined by titrating, which

involves adding incremental volumes of a

known base to an acid, or vica versa.

• If you take acetic acid and slowly add

NaOH, you can get a pH curve (titration

curve)

The titration curve of acetic acid. The molecular species that

predominate at low (acetic acid) and high pH (acetate) are shown. At low

pH (high [H+]), the molecule is protonated and has zero charge. As alkali

is added, [H+] decreases (H+ + OH– → H2O), acetic acid dissociates, and

the carboxyl group becomes negatively charged.

(example for

better

understanding)

ACID-BASE BALANCE IN ECF

(BLOOD)

• Blood [H+]=0.0000004=4x10-8

• Blood pH is normally (-log 4x10-8)

• = 7.4.

• If it changes more than 0.4 (7.0 or 7.8), can’t

easily survive.

• No more physiological: under 7.35

above 7.45

• Between 7.35 and 7.0 is acidosis

• Between 7.45 and 7.8 is alkalosis

REGULATION OF ACID-BASE

BALANCE• Acid-base balance is regulated by:

• acid-base buffer systems,

• Respiratory centers in the brain stem and

• the kidneys,

• The animal may help the rebuilding of the acid-

base balance by increased excretion of [H+] in

the kidneys (in the case of acidosis) or by

increased resorption of [H+] through the kidneys

(in the case of alkalosis).

ACID-BASE BUFFER SYSTEMS ARE

PRESENT IN ALL BODY FLUIDS

• Buffer systems or buffer pairs tend to resist change in pH, when H+ or OH- is added.

• They are weak acids + their salts or weak basis + their salts.

• Buffer systems take up H+ as fluid acidity increases, and give down H + as fluid acidity decreases (or as alkalinity increases).

• They are meant to PREVENT shifts in the hydrogen ion concentration, and thus the pH. They help minimize such naturally occurring changes.

The weak acid of the buffer system dissociates

slightly: the concentration of the dissociated

product is neglible. The salt dissociates almost

completely.

• http://www.google.hu/imgres?q=buffer+system&hl=hu&sa=G&gbv=2&tbm=isch&tbnid=Xx81qpDG17IswM:&imgrefurl=http://www.biog1105-

1106.org/demos/105/unit1/buffers.html&docid=WYaaSZDymMfK-

M&w=800&h=388&ei=I4ZvTryhOsfE4gS3vMmECQ&zoom=1&iact=hc&vpx=277&vpy=112&dur=29047&hovh=156&hovw=323&tx=179&ty=109&page=1

&tbnh=100&tbnw=206&start=0&ndsp=20&ved=1t:429,r:1,s:0&biw=1151&bih=746

Buffer systems

• Buffer capacity is a measure of the

efficiency of a buffer in resisting changes

in pH. Conventionally, the buffer capacity

is expressed as the amount (mol) of strong

acid or base, that must be added to 1 liter

of the solution to change its pH by one

unit.

• The acidic components of the buffer systems give

down H+ and the basic components bind H+.

BICARBONATE BUFFER

SYSTEM: MAINLY IN ECF• Chemicals involved: carbonic acid (H2CO3)

and sodium bicarbonate (NaHCO3)

• Reactions: HCl + NaHCO3 → H2CO3 + NaCl(weaker acid + salt) NaOH + H2CO3 → NaHCO3 + H2O (weaker base+ water)

• pKa=6.1

• pH=6.1+log[HCO3-]/[ H2CO3]

• in the blood the ration of [HCO3-]/[ H2CO3]=20/1,

• So: pH=6.1+log20/1= 6.1+1.3=7.4

H2CO3 can be exreted by the lung in form of CO2

PHOSPHATE BUFFER

SYSTEM: MAINLY IN ICF

• Especially important in nephrons where it controls the pH of tubular fluid and urine.Chemicals involved: dihydrogen phosphate - H2PO4-

and monohydrogen phosphate - HPO42-

• Reactions: HCl + Na2HPO4 → NaH2PO4 + NaCl NaOH + NaH2PO4 → Na2HPO4 + H2O

• pKa =6.8

• pH=6.8+log[HPO42-]/[H2PO4

-] in the blood the ration of [HPO4

2-]/[H2PO4-]=4/1,

• So: pH=6.8+log4/1=6.8+0.6=7.4

PROTEIN BUFFER SYSTEM: IN

PLASMA AND ICF

• This one involves plasma proteins and various proteins inside the cells, including hemoglobin. Recall that proteins are made up of amino acids. The acid-base behavior of native intact globular protein is determined by the ionizable groups of R and the -amino and -carboxyl groups at the end.

• pKa of the proteins=4.9-6.4

HEMOGLOBIN BUFFER

SYSTEM• Hemoglobin buffer system Although there is a

relatively low concentration of hemoglobin in the blood it functions as an effective buffer because it has several buffering groups per molecule. The various buffering groups have different pKa values (6.5-7.8), so buffering by hemoglobin cannot be characterized by a single Henderson-Hasselbalch equation. The numerus imidazole groups (8% histidine in hemoglobin), with pKa values of approximately 7, are responsible for much of the buffering carried out by hemoglobin in the blood. Oxygenated haemoglobin (H:Hb02, pKa:6.6) is a stronger acid than deoxygenated haemoglobin (H:Hb, pKa:6.8). This means that oxygenation of a haemoglobin solution will lead to a decrease in pH or it can be put the opposite way - release of O2 promotes uptake of protons.

•