Isabel Regoli

Biology 120

EXP 2 ADAPTATION Short lab write up

02/09/2017

ABSTRACT

Pacific marine teleost fish live in different water temperature ranges, with different body temperature (T b), yet they

live similar lifestyles. Temperature affects animal physiology, fish may have evolved and adapted metabolic

enzymes to keep their swimming performance elevated, specifically lactate dehydrogenase (LDH )for burst

swimming. In this study, we look at how thermal stress affects enzyme activity, looking at binding affinity, rate of

reaction, and thermal stability in seven different species of fish. The fish studied are: Solea solea (Common sole),

Hippoglosssus stenolepis (Alaskan halibut), Gadus microcephalus (True cod), Dissostichus eleginoides (Pacific

seabass), Ophiodon elongates (Pacific lingcod), Thunnus albacares (Ahi tuna), and Xiphias gladius (Pacific

swordfish). These fish are adequate for this study because of the different T b they have, which respectively are 18-

19oC, 5.4-11.8 oC, 4-11 oC, 2-11 oC, 6-9 oC, 25-30oC, and 20-35 oC. After retrieving the fish from Whole Foods and

measuring protein concentration, the activity of the enzyme was measured through an activity assay with excess

pyruvate. Km and Vmax were measured using the Lineweaver-Burke plot at both 25 oC and 35 oC. Fish homogenate

was then used to measure thermal stability at both temperatures. Results showed decrease in LDH affinity as

temperature increases, suggesting that higher temperatures alter its binding abilities. The results from Vmax suggest

that increasing the temperature makes LDH consumes pyruvate in less time. The results can suggest that fish with

higher burst swimming abilities have lower K m. In addition, endothermic fish, such as tunas, will have higher V max.

The thermal stability results show that as time passes, enzyme activity decrease. In species that are highly active,

such as the Ahi tuna, enzyme stability decreases rapidly, whereas stationary fish consume less LDH over the same

period of time.
RESULTS

see that Km values measured at 35oC appear to be higher for all seven species. We can expect a higher affinity in fish

that are more stationary, and don’t need high amounts of pyruvate to be converted into lactate. Therefore, fish such

the Dover Sole, which have limited locomotion, will have higher Km values than fish such as Yellowfin tuna or

swordfish, which need to produce much energy to be very active. The more concentrated the substrate, the more

likely it is that it will bond to LDH. Fish such as the seabass, lingcod, or true cod might expect higher K m values due

to the sedimentary life style they have as bottom-dwellers. Organisms living in perpetually cold environments, such

as the Alaskan halibut, Pacific seabass, or Pacific lingcod, usually have enzymes that have adapted and work well

even with low-substrate affinity, therefore will usually have higher K m at low temperatures.

When measured at 35oC, all Vmax values increase, compared to the 25oC treatment. This means that, at higher

temperatures LDH converts more pyruvate into lactate. This is expected since at higher temperatures molecules

move faster, and therefore have a higher probability of coming into contact and reacting. This also goes along with

the idea the two fish with the highest body temperatures, the Ahi tuna and Pacific swordfish, have some of the

highest Vmax. In addition, the Vmax looks at the amount of protein enzyme per gram of tissue in each fish. This means

that the Ahi tuna and the Pacific swordfish have the most enzyme per gram of tissue, probably due to the fact that

they are extremely active fish, unlike the common sole which is a sedimentary species.

As time progresses, we can observe for most species that LDH activity is decreasing, due to the fact that it is

consuming all the available pyruvate. Common sole, Pacific seabass, and Pacific swordfish have low R2 values, so

the data and outcomes cannot be considered very reliable. Ahi tuna seems to be the fish with the most enzyme

activity, probably due to its high activity, but oddly the swordfish seems to have close to no activity at all. The cod,

lingcod, sole, and halibut seem to fall in the middle. For the sole, this could be due to its sedimentary life style that

doesn’t require high enzyme activity, for the other three, this could be due to the cold waters they live in and the

way the regulate their enzyme activity to survive.

FIGURES

0.80

0.70

0.60

0.50
Km (mM)

0.40

0.30

0.20

0.10

0.00
Common Sole Alaskan Halibut True Cod Pacific Seabass Pacific Lingcod Ahi Tuna Pacific
Swordfish
Fish species

Figure 1. LDH concentration observed at half the maximal velocity at two temperatures. In black are the Km
values observed at 25oC, while in grey are the Km values at 35oC. Km found through the equation of 1/V vs 1/[S]
best fit line using Km = -1/x-intercept. N=1 for each of the seven species looked at. Habitat temperature for the
common sole is typically 8-24oC, for the Alaskan halibut 3-8 oC, for the true cod 0-15 oC, for the Pacific seabass -2-
10 oC, for the Pacific lingcod 6-9 oC, for the Ahi tuna 18-31 oC, and for the Pacific swordfish 25-30 oC.
Table 1. Maximum rate of LDH consumption in seven Eastern Pacific fish species. In order to calculated Vmax,
the raw Vmax was multiplied by the homogenization dilution and by the assay dilution, different in each species of
fish. Using the Lambert-Beer equation, his number, the absorbance, was then divided by ɛ*l. The extinction
coefficient, ɛ, was equal to 6.22 absorbance units/mmol of NADH, while the length for this assay was equal to 0.01
m. Listed are also the predicted body temperatures of each species.

Species Vmax 25oC Vmax 35oC Predicted Tb

(IU/g) (IU/g) (oC)
Common sole 261 495 18.8
Alaskan Halibut 618 1306 5.4-11.8
True cod 694 1194 4-11
Pacific Seabass 1199 2190 2-11
Pacific lingcod 1578 3064 6-9
Ahi tuna 1875 2686 25-30
Pacific Swordfish 2250 4913 20-35
 0.5

Time (min)
0
Log of Residual Activity

-0.5

-1

-1.5

-2

-2.5

Figure 2. Thermal stability of seven different species of fish. Logarithm of base 10 was taken of the percent
residual activity of LDH of each species. Lines represent treadlines of all data per each fish. The gray solid line
represents the common sole, the black dashed line the Pacific seabass, the blue dashed line the Pacific swordfish, the
blue solid line the Pacific lingcod, the red dashed line the Alaskan halibut, the black solid line the true cod, and the
gray dashed line the Ahi tuna. N=1 for each species. Common sole has an equation of y = -0.0473x – 0.052 with
R2 = 0.3233. Pacific seabass has an equation of y = -0.0195x + 0.0229 with R2 = 0.9118. Pacific swordfish has an
equation of y = -0.0035x – 0.0224 with R2 = 0.0266. The Pacific lingcod has an equation of y = -0.0361x – 0.0134
with R2 = 0.9835. The Alaskan halibut has an equation of y = -0.0439x + 0.024 with R2 = 0.9356. The true cod has
y = -0.0413x – 0.0301 with R2 = 0.9879. The Ahi tuna has an equation of y = -0.0976x – 0.0197 with R2 = 0.9828.