Physical properties of proteins

• Protein solutions exhibit colloidal properties – they

scatter light and exert osmotic pressure
• Molecular weights vary – insulin (5700Da),
hemoglobin (68000Da), albumin(69000Da)
• Shape of proteins varies –globular(insulin),
oval(albumin), elongated(fibrinogen)
• Isoelectric pH- amphoteric nature
• Denaturation
• Ion binding capacity
• solubility
 Precipitation reactions of proteins
• Purification of enzymes and other proteins usually
start with precipitating them from solution
• The stability of proteins in solution will depend
mainly on the charge and hydration. Polar groups of
proteins tend to attract water molecules around
them to produce shell of hydration.
• Any factor which neutralises the charge and removes
the shell of hydration will cause precipitation of
proteins
Procedures used for protein precipitation

• Salting out
- when a neutral salt such as ammonium sulfate
or sodium sulfate is added to protein solution
the protein shell is removed and protein is
pptd.
- higher the molecular weight of protein, salt
required for precipitation is lesser
• Isoelectric precipitation
• Precipitation by organic solvents
• Precipitation by heavy metals
• Precipitation by alkaloidal reagents
Urea Cycle

Dr.Divya dharshini
Dept of biochemistry
 Urea cycle
Urea biosynthesis occurs in four stages:

(1) Transamination
(2) Oxidative deamination of glutamate
(3) Ammonia transport
(4) Reactions of the urea cycle.

Also called as Krebs-Henseleit cycle or Ornithine cycle

First metabolic pathway to be elucidated(1932).

Site of Urea Cycle : In Liver partially mitochondria and cytosol.

Transamination Aminotransferase
PLP

Oxidative
deamination

Urea cycle
 Transamination
• Transamination: Transfer of amino group to α-
ketoglutarate. There are several
aminotransferases specific to different amino
acids.
 Oxidative deamination

•The amino group of glutamate is released as ammonia, regenerating α

-ketoglutarate, by an enzyme glutamate dehydrogenase.
•Glutamate dehydrogenase requires NAD+ or NADP+ as cofactor. This is the
only enzyme known that has specificity for both type of cofactors.
•This enzyme is allosterically inhibited by GTP and activated by ADP.
 Excretory forms of nitrogen

a) Excess NH4+ is excreted as ammonia (microbes, aquatic

vertebrates or larvae of amphibia),
b) Urea (many terrestrial vertebrates)
c) or uric acid (birds and terrestrial reptiles)
 The amino group is taken to liver for nitrogen
excretion

Glutamate releases its amino group

as ammonia in the liver.

The amino groups from many of the

Glutamate a-amino acids are collected in the
dehydrogenase
liver in the form of the amino group
of L-glutamate molecules.

The glutamate dehydrogenase of mammalian

liver has the unusual capacity to use either
NAD+ or NADP+ as cofactor
 Sources of ammonia for the urea cycle:
• Oxidative deamination of Glutamate, accumulated in
the liver by the action of transaminases and
glutaminase.

• Glutaminase reaction releases NH4 that enters the urea

cycle in the liver (in the kidney, it is excreted into the
urine)

• Catabolism of Serine, Threonine and Histidine

(nonoxidative deamination) also releases ammonia.
 Ammonia has to be eliminated
• Ammonia is toxic, especially for the CNS, because it
reacts with -ketoglutarate, thus making it limiting
for the TCA cycle  decrease in the ATP level

• Liver damage or metabolic disorders associated with

elevated ammonia can lead to tremor, slurred speech,
blurred vision, coma, and death
• Normal conc. of ammonia in blood: <40 mg/dl
Entry of nitrogen to mitochondria
Overview of Urea cycle
Step1: Formation of carbamoyl phosphate

Carbamoyl phosphate synthase I

CO2 + NH4 + Carbamoyl phosphate
N-acetyl
glutamate
2 Mg-ATP + H2O 2 Mg-ADP+Pi
Step2 : Formation of Citrulline
Step3: Formation of Arginosuccinate
Step4: Formation of Arginine
 Urea Cycle
Step 5: Formation of Urea

1
ENERGETICS

• 2 ATPs are utilized for the synthesis of carbamoyl

phosphate.
• 1 ATP is converted to AMP and PPi to produce
Arginosuccinate which is equals to 2 ATP
Net utilization = 4 ATP

• NH4+ + CO2 + aspartate + 3 ATP --->

urea + fumarate + 2 ADP + AMP + 4 Pi
 Regulation
• Coarse regulation:
The enzyme level changes with the protein
content of diet. During starvation, the activity of urea
cycle enzymes is elevated to meet the increased rate
of protein catabolism.
• Fine regulation:
The major step is catalyzed by Carbamoyl
phosphate-I, where the positive effector is N-acetyl
gultamate(NAG).It is formed from the glutamate and
acetyl coA. Arginine is the activator of NAG synthase.
• Compartmentalization:
2 enzymes are in mitochondrial matrix
and rest in cytosol. The inhibitory effect of
fumarate on its own formation is minimized.
N-acetylglutamic acid – allosteric
activator of CPS-I

• High concentration of Arg →

stimulation of N-acetylation of
glutamate by acetyl-CoA
Disorders of Urea Cycle
S.No Disorder Enzyme involved

1. Hyperammonemia type I Carbamoyl phosphate synthase I

2. Hyperammonemia type II Ornithine transcarbamoylase

3. Citrllinemia Arginosuccinate synthase

4. Arginosuccinicaciduria Arginosuccinase

5. Hyperargininemia Arginase
Deficiencies of urea cycle
enzymes
•Hereditary deficiency of any of the Urea Cycle
enzymes leads to hyperammonemia - elevated
[ammonia] in blood.
•Elevated ammonia is toxic, especially to the brain.
•The clinical symptoms: Vomiting, lethargy,
irritability, ataxia, and metal retardation.
•If not treated immediately after birth, severe
mental retardation results.
•Total lack of any Urea Cycle enzyme is lethal.
 Ammonia toxicity
Ammonia encephalopathy

• Increased concentration of ammonia in the blood and other biological fluids

→ ammonia diffuses into cells, across blood brain barrier → increased
synthesis of glutamate from a-ketoglutarate, increased synthesis of
glutamine
 alpha-ketoglutarate is depleted from CNS → inhibition of TCA cycle and
production of ATP
• Neurotransmitters – glutamate (excitatory neurotransmitter) and GABA
(inhibitory neurotransmitter)may contribute to the CNS effects.
 N-acetylglutamate synthase deficiency:

• Deficiency or genetic mutation of enzyme (autosomal recessive) → urea

cycle failure.
• A severe neonatal disorder with fatal consequences, if not detected
immediately upon birth.
• Hyperammonemia and general hyperaminoacidemia in a newborn (liver
contain no detectable ability to synthesize N-acetylglutamate).
• Early symptoms include lethargy, vomiting, and deep coma.
• Treatment with structural analog N-carbamoyl-L-glutamate – activates CPS-
I, mitigates the intensity of the disorder,
 Carbamoyl phosphate synthetase (CPS I) deficiency:

• Autosomal recessive metabolic disorder, associated with mental retardation

and developmental delay.
• Hyperammonemia has been observed in 0 – 50% of normal level of CPS-I
synthesis in the liver.

Ornithine transcarbamoylase (OTC) deficiency

• The most common urea cycle disorder, resulting in a mutated and ineffective
form of the enzyme.
• X-linked recessive disorder caused by a number of different mutations in the
OTC gene – males are generally more seriously affected than females (males
are asymptomatic as heterozygotes).
• Complications with OTC may include mental retardation and developmental
delay.
 Argininosuccinate synthase deficiency – citrullinemia (citrullinuria)

• Autosomal recessive metabolic disorder, inability to condense citrulline with

aspartate.
• Accumulation of citrulline in blood and excretion in the urine.
• Type I citrullinemia - usually becomes evident in the first few days of life.
• Type II citrullinemia - the signs and symptoms usually appear during adulthood
and mainly affect the nervous system.
• Therapy – specific supplementation with arginine for protein synthesis and for
formation of creatin and ornithin.
 Argininosuccinate lyase deficiency (argininosuccinate aciduria)

• Rare autosomal recessive disorder, argininosuccinate is excreted in large amount in

urine.
• The severity of symptoms varies greatly, it is hard to evaluate the effect of therapy –
useful is dietary restriction of nitrogen.

Arginase deficiency (argininemia)

• Rare autosomal recessive disorder that cause many abnormalities in development

and function of CNS.
• Accumulation and excretion of arginine in urine and arginine precursors and
products of arginine metabolism.
• Therapy – low nitrogen compounds diet (including essential amino acids
 Objectives
• Creatinine
– Importance
– Formation
– Normal blood & urine levels
– Alterations in diseases
 Creatine Phosphate

• Creatine is present in muscle, brain, blood etc. as

phosphocreatine, a high energy compound.

• MW 113 D

• CrP can be rapidly mobilized to maintain the

intracellular level of ATP during the first few minutes
of intense muscular contraction.
 Muscle contraction
ATP ADP +Pi
Creatine kinase

NH NH  P
HN= C HN= C
N CH2 COO - N CH2 COO -
CH3 CH3
Creatine Creatine Phosphate

Estimation of Creatine kinase helps in the diagnosis of AMI and

skeletal muscle injury
 Creatine

Synthesized from three amino acids –

Glycine, Arginine & Methionine
 BIOSYNTHESIS OF CREATININE
L-Arginine
NH2 Glycocyamine
KIDNEY [Guanidoacetate]
H2N+ = C Glycine Arginine- Glycine NH2
NH Transamidinase
H2N+ = C
CH2 Ornithine
NH CH2 COO -
CH2
SAM
CH2 LIVER Guanidoacetate
S-adenosyl Methyl Transferase
H - C - NH3+
homocysteine
COO -

NH
HN= C
N CH2 COO -
CH3 Creatine
 BIOSYNTHESIS OF CREATININE

NH
HN= C
N CH2 COO -
CH3 Creatine
ATP
MUSCLE
& BRAIN
Non enzymatic ADP
H O NH  P
N C= Non enzymatic HN= C
HN = C
N CH2 Pi + H2O N CH2 COO -
Creatinine MUSCLE & BRAIN CH3 Creatine PO4
CH3
 Degradation of Creatine

• Creatine and creatine phosphate spontaneously

cyclize at a slow constant rate to form creatinine,
which is excreted in urine.

• Creatinine is normally rapidly removed from the

blood by the kidneys. A rise in blood creatinine is a
sensitive indicator of kidney dysfunction.
 CH3
Creatine kinase

ATP ADP
Creatine Creatine Phosphate

CH3 Pi + H2O
H2O

Creatinine
 Creatinine in kidney
• Creatinine is completely filtered by the glomerulus.

• 10 – 15 % is actively secreted in PCT.

• Creatinine clearance reflects GFR.

• In case of severe renal dysfunction, creatinine clearance

rate will be overestimated. Because the active secretion of
creatinine will account for a larger fraction of the total
creatinine cleared.
 • Serum Creatinine level :
Men : 0.7 to 1.3 mg / dl.
Women : 0.6 to 1.1 mg / dl.

• Creatinine excretion in urine :

1.0 to 1.6 gm/day.

• Men tend to have higher levels of creatinine because of

more skeletal muscle mass than women.

• Vegetarians tend to have lower creatinine levels,

because vegetables contain no creatine.
 Increased serum creatinine in
• Impaired kidney function
• Massive rhabdomyolysis/ crush injury
• A lot of meat in diet
• Athletes taking oral creatine
• Hyperthyroidism
• A very large muscle mass
– body builders, anabolic steroid users, acromegaly
• Drugs – block tubular secretion of creatinine
– probenecid, cimetidine, triamterene, trimethoprim,
amiloride.
THANK YOU